Gene of rice dihydrodipicolinate synthase and DNA relating to the same

ABSTRACT

In accordance with this invention, there is provided a DNA sequence which can encode an enzyme protein functioning as the rice dihydrodipicolinate synthase (DHDPS). One example of the DNA sequence according to this invention is the DNA having the nucleotide sequence shown in SEQ ID No. 1 of Sequence Listing. Additionally, this invention provides the DNA having the nucleotide sequence of SEQ ID No. 5, as well as the DNA having the nucleotide sequence of SEQ ID No. 7 of Sequence Listing, as the DNA which is capable of encoding the protein having the DHDPS activity. When cultivation is made of the plant cells in which there has been introduced a recombinant vector having carried therein a DNA fragment containing the DNA of this invention as inserted at downstream of the promoter of said recombinant vector, it is feasible to regenerate from said plant cells a transgenic plant having a high lysine content.

FIELD OF THE INVENTION

The present invention relates to a gene capable of encoding thedihydrodipicolinate synthase of rice (Oryza sativa), and also to a DNAwhich relates to said gene. More specifically, this invention relates tonovel DNAs for encoding such dihydrodipicolinate synthase of rice whichparticipates in the lysine biosynthetic pathway in rice plant.Additionally, the invention relates to DNAs for encoding novel proteinshaving the activity of the dihydrodipicolinate synthase. Stilladditionally, the invention encompasses Escherichia coli as transformedby introduction of said novel DNA, as well as such transgenic plants astransformed by said novel DNA, and the seeds of such transgenic plants.

Still more additionally, this invention encompasses novel recombinantvectors in which said novel DNA has been inserted.

BACKGROUND ART

The grain seeds of cereal plants such as rice, corn and wheat areimportant nutritious sources for humans, cattle and others. However,these grain seeds are nutritionally of a more or less poor value,because of their low contents of lysine as one of the essential aminoacids. It has been desired to create a new variety of plant which iscapable of generating a cereal seed with a high lysine content and of ahigh nutritional value.

It has been known that, in a diaminopimelic acid-producing pathway ofthe lysine biosynthetic pathway in plants, a condensation binding takesplace between aspartic acid-β-semialdehyde and pyruvic acid under theaction of the dihydrodipicolinate synthase (sometimes abbreviated as“DHDPS” hereinafter) to produce 2,3-dihydropicolinic acid, from whichdiaminopimelic acid is then produced by enzymatic reactions through 5steps, and that lysine is produced from the diaminopimelic acid asproduced (Experimental Lecture Series of Biochemistry, Vol. 11, pp.511-517, Tokyo Chemical Dojin). It is known that DHDPS is an enzymeprotein having an activity to produce 2,3-dihydropicolinic acid byinvolving the condensation binding between aspartic acid-β-semialdehydeand pyruvic acid.

A report tells that the lysine content in the seed of a useful plantsuch as corn (Zea mays), tobacco (Nicotiana tabacum), rape seed(Brassica campesteris) and soybean (Glycine max) can be enhanced byintroduction of the gene for encoding DHDPS, that is, the DHDPS gene,into the plant and then expressing the function of said gene in thetransformed plant (PCT International Publication WO 95/15392,JP-W-7-504821, and “Biotechnology”, Vol. 13, pp. 577-582, 1995).

The DHDPS genes have been isolated already from the plants of wheat,corn, soybean and tobacco. The nucleotide sequences of the DNA of theseDHDPS genes have been elucidated. For example, the following techniquesare reported in a number of literatures.

(1) The analysis of the amino acid sequence of wheat DHDPS, and theisolation of the DHDPS gene from wheat (JP-A-3-127984).

(2) The isolation of DHDPS gene from corn, and the nucleotide sequencingof the DNA of said gene (Molecular & General Genetics, Vol. 228, pp.287-293, 1991).

(3) Transformation of corn by means of a DNA as obtained by amodification of the DNA of the DHDPS gene of corn, and theover-production of the lysine content in the corn seed (U.S. Pat. No.5,545,545).

(4) A DNA as obtained by such a modification of the DNA of the tobaccoDHDPS gene that a transgenic plant as transformed by the modified DNA ismade free from the sensitivity of the DNA-encoded DHDPS to the feed-backinhibition by lysine, so that the lysine content in the transgenic plantcan be over-produced (The Plant Journal, Vol. 8, No. 5, pp. 733-743,1995).

(5) Soybean DHDPS gene (Plant Molecular Biology, Vol. 26, pp. 989-993,1994).

(6) A method for over-producing the lysine content in a plant seed,which comprises transformation of the plant with using a DHDPS gene asderived from a bacterial species, for example, Escherichia coli(European Patent Application Publication No. 0 485 970 A2).

To the best of the knowledge of the present inventors, no literatureshave been known, which report an analysis of the amino acid sequence ofthe rice DHDPS or which report a recovery of the gene of the rice DHDPSor the utilization of the rice DHDPS gene.

It is an object of this invention to provide the rice DHDPS gene whichis produced from the rice plant. It is another object of this inventionto provide several novel DNAs for encoding a protein having the riceDHDPS activity. It is still another object of this invention totransform useful plants such as corn, rice, soybean, wheat and barley byusing said novel DNA for encoding a protein having the DHDPS activity,and to provide such a novel transgenic variety of a useful plant whichhas gained an ability to generate a seed of a high lysine content.

Any further objects of this invention will be apparent in the followingdescriptions.

DISCLOSURE OF THE INVENTION

In order to attain the above described various objects, the presentinventors have now made a series of investigations. A firstinvestigation has been carried out in order to produce the DHDPS genefrom the rice plant. To obtain consequence of the investigations, thepresent inventors have conducted some experiments wherein the total RNAwas extracted from a young rice plant, for example, disrupted green stemand leave, by a method known in the genetic engineering technology; andwherein from the so extracted total RNA was then isolated mRNA by aconventional method; and wherein from said mRNA were successfullyproduced cDNAs by a commercially available cDNA synthesis kit. It hasnow been found from the results of a great number of the inventor'sempirical experiments that, when the resulting cDNAs mentioned above areconjugated to a such phage vector which had been prepared by treatingthe EcoRI cleavage end of a DNA fragment of a phage vector λgt11(commercially available from STRATAGEN, LTD.) with a calf smallintestine-derived alkaline phosphatase, there can be produced replicablerecombinant λ phages.

It has now further been found that when Escherichia coli Y1088 strain isinfected with said recombinant λ phages and then incubated, there can beproduced a large number of recombinant λ phages in the forms of numerousplaques comprising the lysogenized bacteria cells; and that the numerousrecombinant λ phages present in the resulting numerous plaques arecomposed of a wide variety of the phages containing therein theaforesaid rice-derived cDNAs and can thus be utilized as a cDNA libraryof rice.

With reference to the amino acid sequence of the wheat DHDPS protein asdescribed in JP-A-3-127984 and also to one speculative nucleotidesequence of the wheat DHDPS gene, as well as to the nucleotide sequenceof the corn DHDPS gene as described in the literature “Molecular &General Genetics”, Vol. 228, pp. 287-293 (1991), the present inventorshave now chemically synthesized a first oligonucleotide comprising 25nucleotides, as well as a second oligonucleotide comprising 24nucleotides, which are both believed to be appropriate to be used asprimers for PCR method.

When a mixture of the aforesaid rice cDNA library (namely, theabove-mentioned recombinant λ phages) with the first oligonucleotide andthe second oligonucleotide as above has been used to conductamplification of DNA by PCR method, it has now been found that the firstand second oligo-nucleotides can serve as the primers (the complementaryDNA) which to be required for the PCR method, and that the cDNAs presentin the rice cDNA library can serve as the template, so that a part ofthe rice DHDPS gene-derived cDNA can be amplified. The present inventorscan have then successfully produced an amplified product of said part ofthe rice DHDPS gene-derived cDNA, as a probe DNA, from the resulting PCRamplification mixture.

The present inventors have used the so produced probe DNA as a screeningmaterial to carry out the phage plaque hybridization method, and thenthere has been got a success fortunately to isolate such four plaquescomprising the recombinant λ phages having integrated with the said riceDHDPS gene, from said previously produced rice cDNA library (composed of300,000 plaques comprising said recombinant λ phages), as results ofnumerous empirical experimental procedures through try and error. Thethus isolated four plaques comprising the recombinant λ phages have nowbeen separately amplified, and the present inventors have isolated andproduced the four types of the individual λ phage DNAs by a conventionalmethod. The four types of the phage DNA thus produced have then beenindividually cleaved with a restriction endonuclease EcoRI to produceDNA fragments. When making comparison of the resulting DNA fragmentswith each other, it has been indicated that these DNA fragments have thesame nucleotide sequence. From further comparison of said DNA fragmentsas produced with the known DNA sequences of the DNA segments of thewheat DHDPS gene and corn DHDPS gene, the present inventors can haveconcluded to approve that the said DNA fragments contain the DNAsequence which is corresponding to the rice DHDPS gene.

The said produced DNA fragments, which are now found to contain such DNAsequence that corresponds to the rice DHDPS gene, have been cleaved witha restriction endonuclease EcoRI. Then, the resultant DNA fragmentproduced by this cleavage has been inserted into and ligated to theEcoRI cleavage site of a commercially available, known plasmid vectorpBluescript II SK(+), by using a DNA ligation kit. The recombinantplasmid vector so produced can be used to transform Escherichia coliXL1-Blue MRF′ strain. The resulting Escherichia coli transformant cellscan be incubated to produce a great number of copies of the bacterialcells. From these bacterial cells so copied is produced the plasmid.Then, a DNA fragment which contains the rice-derived DNA sequencetherein is cleaved out of the DNA of the so produced plasmid, by usingthe restriction enzymes. The DNA fragment as obtained by this cleavageis then subjected to DNA sequencing, whereby it has been confirmed thatthe nucleotide sequence of the DNA sequence, which is inserted in thelatter DNA fragment and which has been approved to correspond to therice DHDPS gene, just exhibits a nucleotide sequence which is describedin SEQ ID No. 1 of Sequence Listing given hereinafter.

To the best of the knowledge of the inventors, it has also been foundthat the DNA having the nucleotide sequence shown in SEQ ID No. 1 ofSequence Listing is a novel DNA which is never described in anyliterature.

It is considered that the protein as encoded by the DNA having thenucleotide sequence of SEQ ID No. 1 is a protein having the amino acidsequence shown in SEQ ID No. 2 of Sequence Listing, and that the saidprotein constitutes the rice DHDPS.

In a first aspect of the invention, therefore, there is provided a DNAfor encoding the rice dihydrodipicolinate synthase which has the aminoacid sequence shown in SEQ ID No. 2 of Sequence Listing hereinafter.

More specifically, the DNA according to the first aspect of theinvention may be a DNA having the nucleotide sequence shown in SEQ IDNo. 1 of Sequence Listing.

The DNA according to the first aspect of the invention is a DNA whichencodes the protein of the rice DHDPS. As described above, the DNA ofthis invention is produced from the cDNA library of rice by the geneticengineering technique through the investigations of the inventors.Because the nucleotide sequence of the DNA of this invention once hasbeen elucidated as above in accordance with the invention, said DNA canbe produced also by chemical synthesis with reference to the nucleotidesequence of SEQ ID No. 1 of Sequence Listing. By polymerase chainreaction (abbreviated as PCR) or hybridization, it is also possible toproduce the DNA of the first aspect of the invention from the DNAlibrary of the rice chromosome in such a way that either a syntheticoligonucleotide as prepared with reference to the nucleotide sequence ofSEQ ID No. 1 is used as a probe, or said synthetic oligonucleotide isused as a primer.

The method for producing the DNA of the first aspect of the inventionfrom the stem and leave of rice by the genetic engineering technology isschematically described hereinbelow.

(1) Preparation of Rice mRNA and Construction of the cDNA Library ofRice

From various tissues of rice plant, for example, stem and leave, rootand callus of Oryza sativa (rice), preferably green stem and leavethereof, there is extracted a total RNA in a conventional manner. Fromthe so extracted total RNA are then removed contaminants such asprotein. The resulting partially purified total RNA fraction is passedthrough a column of oligo dT cellulose to purify the poly(A)⁺ RNA, sothat the MRNA of rice can be obtained.

Subsequently, the rice cDNAs are synthetically prepared by using saidmRNA of rice by means of a commercially available cDNA synthesis kit.The so synthesized cDNAs are inserted into a phage vector, for example,λgt11 vector or λZAPII vector, to produce a great number of therecombinant phages. With these recombinant phages are then infected thecells of Escherichia coli as host cell, followed by incubation, so thata great number of the recombinant phages is produced in the plaques ofthe lysogeized host cells. A series of these procedures can be practicedby using any commercially available cDNA cloning kit.

Such great number of the recombinant phages as obtained in the plaquesof the lysogenized host cells of Escherichia coli are comprising a widevariety of the phages containing therein the cDNAs as derived from rice,and hence the said recombinant phages can be utilized as the cDNAlibrary of rice.

(2) Construction of Primers for PCR Method

By comparing the known nucleotide sequence of the wheat DHDPS gene (forexample, the DNA sequence as described in JP-A-3-127984) with the knownnucleotide sequence of the corn DHDPS gene (for example, the DNAsequence as described in Molecular & General Genetics, Vol. 228, pp.287-293), the present inventors have now detected that there exists anucleotide sequence which is retained in common to the aforesaid knowntwo nucleotide sequences. With reference to the common nucleotidesequence thus detected, the present inventors have chemicallysynthesized and constructed two types of oligonucleotides (namely, theoligonucleotides of SEQ ID Nos. 3 and 4 described in Sequence Listinghereinafter) to be used as the primers (the complementary DNA) for PCRmethod.

(3) Preparation of Probe DNA

For the end to selectively amplify the desired DNA of encoding the riceDHDPS present within the rice cDNA library which was produced as abovein the form of the plaques containing a great number of the recombinantphages, amplification of the DNA of encoding a part of the rice DHDPSgene is done with utilizing the rice cDNA library as the template,according to the PCR method and with using the said two syntheticoligonucleotide as the primer.

After the PCR amplification is done in repetition, the amplified productof such a DNA fragment, which is the part of the DNA sequencecorresponding to the rice DHDPS gene, is recovered and collected as theprobe DNA from the resultant amplification mixture.

(4) Selection of DNA of the Rice DHDPS Gene from the Rice cDNA Library

Then, several numbers of plaques made of the recombinant phages havingthe inserted DNA sequence, which is wholly corresponding to the targetDHDPS gene of rice, are selected and separated from the great number ofsaid plaques made of the recombinant phages which were previouslyproduced as the rice cDNA library. This selection and separation may bedone according to a phage plaque hybridization method with using thesaid probe DNA as the screening material.

Thereby, a DNA fragment carrying therein the target DNA sequence, whichis corresponding to the DNA of the rice DHDPS gene, can be harvested inthe form of the recombinant phage having the said DNA fragment insertedtherein.

Thus, the recombinant phages provided in the form of the plaques asselected by the aforesaid plaque hybridization method are separated andcollected. Then, the phage DNA is harvested from the so collectedrecombinant phages. By treating the so harvested phage DNA by thedideoxy method and the like, it is feasible to decide the nucleotidesequence of the inserted DNA fragment which has been derived from rice.When the amino acid sequence, which shall be defined by theprotein-encoding region (the open reading frame) of the nucleotidesequence of the said inserted DNA fragment as just derived from rice, iscompared with the known amino acid sequence of the corn DHDPS protein orof the wheat DHDPS protein, followed by determining the presence orabsence of the homology between them, it can be identified whether ornot the said inserted DNA fragment contains the DNA sequence which isjust corresponding to the rice DHDPS gene.

Thus, the above-mentioned, inserted DNA fragment, which has beenidentified to contain therein the DNA sequence just corresponding to therice DHDPS gene, can then be cleaved out and recovered from the phageDNA of the aforesaid recombinant phages which was selected and collectedin the aforementioned method.

(5) Cloning of the cDNA Corresponding to the Rice DHDPS Gene

The DNA, which was produced by cleaving the recombinant phages and wasobtained as the DNA fragment containing therein the DNA sequence of therice DHDPS gene as described above, is then used to construct arecombinant plasmid vector, by inserting and ligating the said DNA asproduced just from the recombinant phages, into the EcoRI cleavage siteof a plasmid vector pBluescript II SK(+). The thus constructedrecombinant plasmid vector is then used to transform Escherichia coliXL1-Blue MRF′. By culturing and proliferating the Escherichia colitransformant cells thus produced, there can be cloned the saidrecombinant plasmid which carries the DNA fragment containing thereinthe inserted DNA sequence of the rice DHDPS gene. In this way, the DNAfragment which contains therein the DNA sequence of the rice DHDPS genecan be cloned.

(6) Sequencing of the Cloned DNA

The so cloned recombinant plasmid is then separated and cut withappropriate restriction enzymes to produce such a DNA fragment whichcontained therein the DNA sequence of the DHDPS gene of rice. Bytreating the so produced DNA fragment with a commercially availablenucleotide sequencing kit, it is possible to determine the nucleotidesequence of the DNA which comprises the DNA sequence just correspondingto the rice DHDPS gene. The thus determined nucleotide sequence of theDNA which encodes the rice DHDPS gene is just as described in SEQ ID No.1 of Sequence Listing.

When the DNA sequence obtained as above by the first aspect of theinvention or a DNA fragment comprising said DNA sequence is used as aprobe, the DNA of the DHDPS gene can be obtained also from thechromosome of rice itself in a conventional manner. The DNA of the DHDPSgene which is so obtained from the rice chromosome itself may possiblycontain an intron. Such DNA having the intron intervened therein is alsoencompassed within the scope of the DNA of the first aspect of theinvention, so far as it is the DNA for encoding the rice DHDPS.

The DNA fragment of the nucleotide sequence, which is described in SEQID No. 1 of Sequence Listing and was prepared in the following Example1, and which contains therein the DNA sequence corresponding to the riceDHDPS gene, is inserted and ligated into a pBluescript II SK(+) plasmidvector to prepare a recombinant plasmid vector. Escherichia coliXL1-Blue MRF′ which has been transformed by introduction of the aboveprepared recombinant plasmid vector, is designated as Escherichia coliDAP8-1 strain and was deposited under Accession No. of FERM P-15906 onOct. 14, 1996 at the National Institute of Bioscience andHuman-Technology, the Agency of Industrial Science and Technology, atTsukuba-City, Ibarak-ken, Japan. Further, the Escherichia coli DAP8-1strain has been deposited at the same institute, under Accession No. ofFERM BP-6310, since Mar. 26, 1998, under the provisions of the BudapestTreaty.

The DNA according to the first aspect of the invention is useful in thatsaid DNA enables a great quantity of the rice DHDPS protein to beproduced by chemical synthesis, when the nucleotide sequence of the DNAas elucidated in accordance with the invention is employed as aguideline. Further, the DNA of this invention can contribute a lot to aprogress of the enzymological investigative works of the rice DHDPSprotein.

Furthermore, a recombinant vector may be constructed when the terminusof either the DNA sequence as provided by the first aspect of theinvention or of an appropriate DNA fragment containing therein said DNAsequence are linked to EcoRI linkers, followed by inserting theresulting linked DNA sequence or DNA fragment into such EcoRI cleavagesite of a commercially available plasmid vector pTV118N (manufactured byTAKARA Shuzo Co., Ltd., Japan), which is present at downstream of theEac promoter of the vector. It is expected that, when Escherichia coliwhich has been transformed with the resulting recombinant vector soconstructed as above is cultured, the transformed cells of Escherichiacoli can intracellularly generate such a protein which has the DHDPSactivity. Thus, a protein having the rice DHDPS activity can beexpectedly produced in a culture of the Escherichia coli cell which hasbeen transformed by insertion of the above recombinant vector such thatthe so transformed Escherichia coli cell can express the DNA of thefirst aspect of the invention.

Meanwhile, it has generally been known widely by a person of an ordinaryskill in the art, that even such an amino acid sequence, which isproduced by a modification of an intact amino acid sequence of a proteinhaving a physiological activity, through a deletion of one or pluralamino acids, and/or through replacement thereof by other amino acids,and/or through addition of one or plural amino acids to the intact aminoacid sequence, is possible to retain the physiological activity of theparent protein having said intact amino acid sequence. The DNA accordingto the first aspect of the invention is able to encode the proteinhaving the DHDPS activity, even when a part or plural parts of thenucleotide sequence of the DNA of the first aspect of this invention hasor have been modified.

In other words, the novel DNA according to the first aspect of theinvention can retain the ability to encode the protein having the DHDPSactivity, even after one or plural nucleotides, for example, 1, 2 or 3to 10 nucleotides present in the nucleotide sequence of the DNA of thefirst aspect of this invention has or have been modified with othernucleotides.

In a second aspect of the invention, therefore, there is provided a DNAfor encoding such a protein which has the dihydrodipicolinate synthaseactivity, and which protein has such an amino acid sequence that isformed by modification of the amino acid sequence shown in SEQ ID No. 2of Sequence Listing, with said modification being made by deletion ofone or plural amino acids from said amino acid sequence of SEQ ID No. 2,and/or by replacement of one or plural amino acid present in said aminoacid sequence by other amino acids, and/or by insertion or addition ofother amino acids to said amino acid sequence.

The DNA of the second aspect of the invention is thus such a DNA whichhas been modified from the DNA of the first aspect of the invention.Thus, the DNA of the second aspect of the invention can be produced bymodifying the nucleotide sequence of the DNA of the first aspect of theinvention, for example, by making a site-directed mutagenesis, in such away that the resulting nucleotide sequence as modified can encode aprotein having such an amino acid sequence which is modified bydeletion, replacement or addition of an amino acid (or amino acids) atspecific site(s) of the protein that is encoded by the DNA of the firstaspect of the invention.

The modified DNA of the second aspect of the invention can be producedalso by subjecting the bacterial cells having a DNA fragment containingtherein the DNA of the first aspect of the invention to a mutationprocess, and subsequently selectively recovering from the resultingmutated cells, for example, a DNA having such a nucleotide sequencewhich is hybridizable with the DNA of the nucleotide sequence of SEQ IDNo. 1 of Sequence Listing under stringent conditions, but which ispartially different from the nucleotide sequence of SEQ ID No. 1. Theterm “stringent conditions” herein referred to does mean such conditionsunder which any hybrid which is specific to the DNA of the first aspectof the invention is formed, while a hybrid which is non-specific to saidDNA is not formed. The stringent conditions can hardly be defined orspecified numerically. However, one typical example of the stringentconditions is such one which can permit the hybridization of two nucleicacids or DNAs to take place at a high homology, for example, at a 98% orhigher homology, but can inhibit the hybridization of two nucleic acidsfrom occurring at a homology of lower than the 98% value.

Thus, the DNA of the second aspect of the invention may be, for example,a DNA which is such a DNA having a nucleotide sequence partiallydifferent from the nucleotide sequence shown in SEQ ID No. 1 of SequenceListing but having a high homology to said nucleotide sequence of SEQ IDNo. 1, and which DNA can hybridize with the DNA having said nucleotidesequence shown in SEQ ID No. 1 of Sequence Listing under stringentconditions, and which DNA encodes the protein having thedihydrodipicolinate synthase activity.

Specifically, the DNA of the second aspect of the invention may be, forexample, the DNA sequence which is designated as a modified DNA-158Nsequence in Example 2 hereinafter and which is prepared according to themethod described in Example 2, as well as the DNA sequence which isdesignated as a modified DNA-166A sequence in Example 3 hereinafter andwhich is prepared according to the method described in Example 3.

The DNA sequence, which is designated as the modified DNA-158N sequence,has the nucleotide sequence of SEQ ID No. 5 of Sequence Listing. Themodified DNA-158N sequence corresponds to such a DNA which is formed bya modifying of the DNA of the nucleotide sequence of SEQ ID No. 1 ofSequence Listing, in such a way that the sequence AAC (a codon forencoding asparagine) present at the positions 472, 473 and 474 of thenucleotide sequence of the DNA of the first aspect of the invention isreplaced by a sequence ATC (a codon for encoding isoleucine) withreplacing the base A (adenine) at the position 473 by a base T(thymine). The modified DNA-158N sequence as above can hybridize withthe DNA of the first aspect of the invention under the stringentconditions.

The protein, which is encoded by the modified DNA-158N sequence, has anamino acid sequence of SEQ ID No. 6 of Sequence Listing and has theDHDPS activity.

The modified DNA-166A sequence mentioned above has the nucleotidesequence of SEQ ID No. 7 of Sequence Listing. The modified DNA-166Asequence corresponds to such a DNA which is formed by modifying of theDNA of the nucleotide sequence of SEQ ID No. 1 of Sequence Listing, insuch a way that the sequence GCA (a codon for encoding alanine) at thepositions 496, 497 and 498 of the nucleotide sequence of the DNA of thefirst aspect of the invention is replaced by a sequence GTA (a codon forencoding valine) with replacing the base C (cytosine) at the position497 by a base T (thymine). The modified DNA-166A sequence as above canhybridize with the DNA of the first aspect of the invention under thestringent conditions.

The protein, which is encoded by the modified DNA-166A sequence, has anamino acid sequence of SEQ ID No. 8 of Sequence Listing and has theDHDPS activity.

Additionally, a further example of the DNA of the second aspect of theinvention may be specifically a DNA sequence having such a nucleotidesequence which is prepared by replacing the adenine at the position 473of by the nucleotide sequence of SEQ ID No. 1 of Sequence Listing bythymine, and also by replacing the cytosine at the position 497 bythymine. This DNA sequence so modified may encode a protein having thedihydrodipicolinate synthase activity, and having such an amino acidsequence which is prepared by replacing the asparagine at the position158 of the amino acid sequence of SEQ ID No. 2 of Sequence Listing byisoleucine and also by replacing the alanine at the position 166 byvaline.

Generally, as the methods for modifying a nucleotide sequence present ina part of a DNA sequence, there are known several methods, including theKunkel method (Methods in Enzymology, Vol. 154, No. 367), and theoligonucleotide-direct dual amber method and other methods.

The essential object of this invention is to create a novel transgenicplant species which is so transformed to generate a seed of a highlysine content. Hence, this invention requires such a modified DHDPSwhich can exhibit an enzyme activity so modified that the resultingenzyme activity of the modified DHDPS is made insensitive to that theDHDPS participating in the lysine bio-synthetic pathway can involve thefeed-back inhibition owing to the bio-product, lysine. As well, thisinvention requires such a DNA which can encode said modified DHDPS. Forthe purpose of preparing from the DNA of the first aspect of theinvention, a novel modified DNA capable of encoding such a novel DHDPSwhich has been so modified that the resultant modified novel DHDPSenzyme is freed from the sensitivity to the feed-back inhibition bylysine, the inventors have now made some investigations at first.Consequently, the inventors have now found that one or two or morebases, which is or are present in partial regions of the nucleotidesequence of encoding the rice DHDPS according to the first aspect of theinvention, can be replaced by other bases by means of a combination of amodified method of the known Kunkel method with a modifiedoligonucleotide-direct dual amber method.

With making reference to the prior art for the modification of the cornDHDPS gene as described in the U.S. Pat. No. 5,545,545, the prior artfor the modification of the tobacco DHDPS gene as described in The PlantJournal, Vol. 8, No. 5, pp. 733-743, and the prior art for the planttransformation with using the bacterial DHDPS gene as described in theEuropean Patent Application 0 485 970 A2, furthermore, the inventorshave now studied and got a speculation that such a modified DNA, whichis prepared either by replacing the base adenine (A) at the position 473by thymine (T) or replacing the cytosine (C) by thymine (T) at theposition 497 of the nucleotide sequence of the DNA of the first aspectof the invention as shown in as SEQ ID No. 1 of Sequence Listing, wouldbe able to encode a novel modified enzyme protein which retains theDHDPS activity, and of which enzyme activity is made insensitive to thefeed-back inhibition by lysine.

Based on the above speculation, the inventors have now made variousinvestigations and empirical experiments at a great number of times.Consequently, the inventors have now found that the undermentionedrecombinant plasmid vector (referred to as “pDAP8-1” hereinafter) issuitable as a starting material for the preparation of the novelmodified DNA as desired above. Said plasmid vector pDAP8-1 has beenprepared by inserting and integrating the aforesaid DNA fragmentcontaining therein the DNA sequence of the rice DHDPS gene as preparedupon the production of the novel DNA of the first aspect of thisinvention [(namely, the DNA fragment of the recombinant λ phage, i.e.the DNA fragment containing the DNA sequence of SEQ ID No. 1 (referredto as DHDPS-DNA-1143 sequence hereinafter)] into the position betweenthe EcoRI cleavage site and the SacI cleavage site of the plasmid vectorpbluescript II SK (+) by using a DNA ligation kit.

Additionally, the inventors have now chemically synthesized five typesof primers suitable for preparing the above-mentioned target novelmodified DNA from the starting recombinant plasmid vector pDAP8-1according to the PCR methods. Thus, the inventors have prepared a primerNo. 3 comprising an oligonucleotide of the nucleotide sequence of SEQ IDNo. 9 of Sequence Listing; a primer No. 4 comprising an oligonucleotideof the nucleotide sequence of SEQ ID No. 10 of Sequence Listing; aprimer FW-1 comprising an oligonucleotide of the nucleotide sequence ofSEQ ID No. 11 of Sequence Listing; a primer RV-1 comprising anoligonucleotide of the nucleotide sequence of SEQ ID No. 12 of SequenceListing; and a primer BS KPN-1 comprising an oligonucleotide of thenucleotide sequence of SEQ ID No. 13 of Sequence Listing.

When the method described below in detail has been done with using theplasmid vector pDAP8-1 and the above-mentioned five types of the primerscomprising the synthetic oligonucleotides, the inventors now can havesuccessfully produced some specific examples of the modified DNA of thesecond aspect of the invention. These specific examples are a DNAfragment containing the DNA of SEQ ID No. 5 of Sequence Listing, namelya DNA fragment containing the modified DNA-158N sequence, as well as aDNA fragment containing the DNA of SEQ ID No. 7 of Sequence Listing,namely a DNA fragment containing the modified DNA-166A sequence.

Not only the DNA of the first aspect of the invention, but also themodified DNA-158N sequence and the modified DNA-166A sequence may beinserted in the recombinant vector and then may be introduced in therice plant according to the known method for integrating an exogenousgene into plants as described hereinafter in Example 4 or 5, so that theintroduced DNA can express in the resulting transgenic plant astransformed.

It is verified that the transgenic plant of rice as transformed by theDNA of the first aspect of the invention or by the modified DNA-158Nsequence or the modified DNA-166A sequence of the second aspect of thisinvention, can generate a rice plant cell or a rice seed having anenhanced lysine content.

Next, a brief description is given of the method for modifying DNA whichcomprises procedures typically illustrated below in Example 2, and whichcan suitably be used for preparing the DNA fragment containing themodified DNA-158N sequence of the second aspect of the invention.

(1) Cloning of the DNA of the First Aspect of the Invention

By inserting and conjugating the DNA fragment containing the DNA havingthe 1143 nucleotides of SEQ ID No. 1 of Sequence Listing of the firstaspect of the invention, (namely the DNA fragment containing theaforesaid DHDPS-DNA-1143 sequence) into the EcoRI cleavage site of thevector pBluescript II SK(+) by means of a DNA ligation kit, there can beproduced the recombinant plasmid vector pDAP8-1. After introduction ofthis plasmid vector pDAP8-1 in the Escherichia coli XL1-Blue MRF′strain, the resulting transformed cells of Escherichia coli areproliferated. From the resulting copies of the transformed Escherichiacoli cells, there can be extracted a great quantity of the recombinantplasmid vector pDAP8-1, according to a conventional method. The plasmidvectors so obtained contain a great number of the copies of the DNAfragment containing the DHDPS-DNA-1143 sequence. Thus, it is confirmedthat the DNA of the first aspect of the invention is cloned as above.

(2) Construction of Primers for PCR Method

Using a DNA synthesizer (Model-391; manufactured by Applied Biosystems,Co.), five types of the oligonucleotides having the following nucleotidesequences are synthetically prepared as the primers.

(a) Primer No. 3 (primer having the following nucleotide sequence asshown in SEQ ID No. 9 of Sequence Listing)

5′-GCCTCTCTTGTTGAGATACTACCTGTGTTGCC-3′

(b) Primer No. 4 (primer having the following nucleotide sequence asshown in SEQ ID No. 10 of Sequence Listing)

5′-GCAAATCCCTGCTCTGTTACATGAATAGCC-3′

(c) Primer No. FW-1 (primer having the following nucleotide sequence asshown in SEQ ID No. 11 of Sequence Listing)

5′-GTAAAACGACGGCCAGTGAG-3′

(d) Primer No. RV-1 (primer having the following nucleotide sequence asshown in SEQ ID No. 12 of Sequence Listing)

5′-GGAAACAGCTATGACCATG-3′

(e) Primer No. BS KPN-1 (primer having the following nucleotide sequenceas shown in SEQ ID No. 13 of Sequence Listing)

5′-TAGGGCGAATTGTGTGTACCG-3′

(3) Amplification and Recovery of the Required DNA Fragment According toPCR Method

For making the amplification of the required DNA fragment, there areconducted two reactions which are included by the first step of PCRmethod and which are namely the following reactions (A) and (B).

The reaction (A) comprises adding the recombinant plasmid vector pDAP8-1for use as the template, as well as the primers FW-1 (the syntheticoligonucleotide of SEQ ID No. 11) and primer No. 3 (the syntheticoligonucleotide of SEQ ID No. 9, of which the GAT at the positions 15 to17 from the 5′ terminus can induce the ATC at the modified part of theDNA-158N sequence) to a conventional PCR reaction mixtutre [containingTris-HCl, MgCl₂, KCl and 4 types of deoxynucleotide phosphates (dNTP)and La Taq DNA polymerase], and then progressing the amplificationreactions therein. By the reaction (A), there can be produced a DNAfragment (referred to as DNA fragment-A) which has a nucleotide sequencecorresponding to a part of the DHDPS-DNA-1143 sequence, through theamplification reactions.

The reaction (B) comprises adding the primer RV-1 (the syntheticoligonucleotide of SEQ ID No. 12) and primer BS KPN-1 (the syntheticoligonucleotide of SEQ ID No. 13), as well as the vector pDAP8-1 as thetemplate to the conventional PCR reaction mixture of the samecomposition as that used in the reaction (A), and subsequentlyprogressing the amplification reactions therein. By the reaction (B),there can be produced a DNA fragment (referred to as DNA-fragment B)which contains a nucleotide sequence corresponding to a part of theDHDPS-DNA-1143 sequence and which further contains an extension parthaving a SacI cleavage site at the 3′ terminus thereof, through theamplification reactions.

The aforementioned amplification reactions by the PCR method can bepracticed by using a commercially available PCR reactor.

After the completion of these amplification reactions, the amplificationreaction solution coming from the reaction (A) is fractionated by alow-melting agarose electrophoresis, followed by cutting a gel bandcontaining the DNA fragment-A of 480 bp (base pair) obtained as anamplified product, from the agarose gel. Additionally, the amplificationreaction solution coming from the reaction (B) is similarly fractionatedby a low-melting agarose electrophoresis, followed by cutting a gel bandcontaining a DNA fragment-B of 1200 bp (base pair) obtained as anamplified product, from the agarose gel.

By purifying these two cut pieces of the gel bands with a DNApurification kit, for example, Geneclean II kit (manufactured byFunakoshi, Co., Ltd.), a purified product of the said DNA fragment-A anda purified product of the said DNA fragment-B are individually produced.

As the second step of the PCR method, there is conducted a process ofpreparing a DNA fragment of 1200 bp which contains therein a DNAsequence of such a nucleotide sequence as formed by replacing theadenine at the position 473 of the nucleotide sequence of SEQ ID No. 1of sequence Listing by thymine (that is, the DNA sequence correspondingto the DNA-158N sequence of the nucleotide sequence of SEQ ID No. 3 asprovided according to the second aspect of the invention), and which DNAfragment of 1200 bp contains an extension part having a KpnI cleavagesite at the 5′ terminus of said DNA sequence and having a SacI cleavagesite at the 3′ terminus thereof. To this end, the purified product ofthe DNA fragment-A (the sequence of 480-bp length) obtained as theamplify-cation product of the reaction (A) and the purified product ofthe DNA fragment-B (the sequence of 1200-bp length) obtained as theamplification product of the reaction (B)(both for use as thetemplates), as well as the primer RV-1 (the synthetic oligonucleotide ofSEQ ID No. 12), and the primer FW-1 (the synthetic oligonucleotide ofSEQ ID No. 11) are added to a conventional PCR reaction mixture[containing Tris-HCl, MgCl₂, KCl and 4 types of deoxynucleotidephosphates (dNTPs) and La Taq DNA polymerase], followed by progressingthe amplification reactions therein. After the completion of thereactions, the resulting reaction solution is fractionated by alow-melting agarose electrophoresis, followed by cutting a gel bandcontaining the objective DNA fragment of 1200 bp (referred to as DNAfragment-C), from the agarose gel.

By purifying the resultant cut gel band with a DNA purification kit, forexample, Geneclean II kit (manufactured by Funakoshi, Co., Ltd.), apurified product of the DNA fragment-C is produced. This DNA fragment-Chas such structure that this fragment carries therein the nucleotidesequence corresponding to the modified DNA-158N sequence of the secondaspect of the invention and also contains an extension part having aKpnI cleavage site at the 5′ terminus of the DNA fragment and anextension part having a SacI cleavage site at the 3′ terminus thereof.

(4) Cloning of the DNA Fragment Containing the Modified DNA-158NSequence

Next, the DNA fragment-C as produced in the above (3) is used to producea DNA fragment which contains the target modified DNA-158A sequence (afirst example of the modified DNA of the second aspect of theinvention).

To this end, first, the extension parts present at the 5′ and 3′ terminiof the above DNA fragment-C are treated with the restrictionendonucleases KpnI and SacI.

Thereby, the DNA fragment-C is cut and divided to produce such a DNAfragment which carries therein the modified DNA-158A sequence, whichcontains the extension part having the SacI cleavage site at the 3′terminus and in which the 5′ terminus starts at ATG.

In this way, a sample of the DNA fragment containing therein the DNAsequence corresponding to the target modified DNA-158A is obtained.Subsequently, the plasmid vector pBluescript II SK(+) is treated withrestriction endonucleases KpnI and SacI, to produce a truncated plasmidwhich has the KpnI cleavage site at the 5′ terminus and has the SacIcleavage site at the 3′ terminus (and which is referred to aspBluescript II SK(+)-KpnI-SacI-truncated plasmid hereinbelow).

This KpnI-SacI-truncated plasmid is then mixed with said sample of theDNA fragment carrying therein the DNA-158N sequence, followed bysubjecting the resultant mixture to a ligation reaction with using a DNAligation kit, whereby there can be produced a recombinant plasmidcontaining the modified DNA-158N sequence (hereinafter referred to aspBluescript-DNA-158N plasmid).

After introducing the so produced recombinant plasmid in Escherichiacoli XL1-Blue MRF′ to effect the transformation thereof, the resultingtransformed cells of Escherichia coli are cultured and proliferated in aliquid culture medium. A vast quantity of the proliferated bacteriacells of the transformed Escherichia coli is obtained, which containscopies of the recombinant plasmid, namely copies of thepBluescript-DNA-158N plasmid. In this way, the modified DNA-158Nsequence can be cloned. From the cultured cells of the transformedEscherichia coli is extracted and harvested the desired plasmidcontaining the modified DNA-158N sequence, according to a routinemethod.

(5) Recovery of the Modified DNA-158N Sequence

The plasmid carrying therein the modified DNA-158N sequence which wasrecovered in the item (4), is treated and digested with restrictionendonucleases XbaI and SacI.

Thus, a digestion solution can be prepared, which contains the DNAfragment carrying therein the modified DNA-158N sequence and furtherhaving the extension parts wherein the nucleotide sequence ATG isprovided at the 5′ terminus adjacent to the XbaI cleavage site, andwherein the SacI cleavage site is provided at the 3′ terminus.

After the fractionation of the digestion solution is made by low-meltingagarose electrophoresis, a gel band containing the said DNA fragment iscut from the agarose gel. The resulting cut piece of the agarose gelband is dissolved in a TE buffer, and the resulting solution issubjected to extraction with phenol. Thus, the said DNA fragment isrecovered into the phenol extract solution. The phenol extract solutioncontaining the said DNA fragment is mixed with an aqueous 3M sodiumacetate solution and ethanol; and the resultant mixture is left to standat 20° C. for about 6 hours, followed by centrifugation at a lowtemperature, to precipitate the DNA fragment. The precipitated DNAfragment is dried under reduced pressure, to afford the DNA fragment inthe form of a powder, which is the fragment containing the targetmodified DNA-158N sequence. This powder of the DNA fragment carryingtherein the modified DNA-158N sequence is soluble in water.

Furthermore, a DNA-modifying method which is preferably applicable tothe preparation of a DNA fragment carrying therein the modified DNA-166Asequence according to the second aspect of the invention and which isillustrated in Example 3 hereinafter, may be practiced in the same wayand by the same procedures as those for the above-mentioned method forpreparing the DNA fragment carrying therein the modified DNA-158Nsequence.

More specifically, the above-mentioned preferable method for preparingthe DNA fragment carrying the modified DNA-166A sequence may comprise afirst step of cloning the DNA sequence, wherein the recombinant vectorpDAP8-1 containing the DNA of the first aspect of the invention (namely,DHDPS-DNA-1143) is introduced into Escherichia coli XL1-Blue MRF′ andthe transformed Escherichia coli cell is proliferated with following thesame procedures as described in the item (1) of the foregoing Section ofexplaining the “Method for preparing the DNA fragment containing themodified DNA-158N sequence”.

A subsequent step of the said preferable method comprises adding thevector pDAP8-1 for use as the template, as well as the primer FW-1prepared as a synthetic oligonucleotide, and the primer No. 4 (thesynthetic oligonucleotide of SEQ ID No. 10, which is capable of inducingthe modified part “GTA” of the modified DNA-166A sequence due to the TAClocated at the positions 18-20 from the 5′ terminus of saidoligonucleotide) to the conventional PCR reaction mixture of the samecomposition as that used in the reaction (A) described in the item (3)of the foregoing Section of explaining “Method for preparing a DNAfragment containing the modified DNA-158N sequence”, and then effectingthe amplification reactions therein. By the amplification reactions,there can be produced such a DNA fragment (referred to as DNAfragment-D) which carries therein a nucleotide sequence present in acertain region of the aforesaid DHDPS-DNA-1143 sequence.

In the very same manner as for the reaction (B) which is effected by theprocedures described in the item (3) of the Section of explaining“Method for preparing a DNA fragment containing the modified DNA-158Nsequence”, there is subsequently conducted a reaction (B) step for PCRmethod. Thereby, the aforesaid DNA fragment-B is produced asamplification product from the reaction (B).

The above amplification solution which contains the DNA fragment-D asproduced by the reaction (A) with using the primers FW-1 and No. 4, isthen fractionated by low-melting agarose electrophoresis. Then, a gelband containing the DNA fragment-D of 480 bp as the amplificationproduct is cut and separated out from the agarose gel. The amplificationsolution containing the DNA fragment-B as produced by the reaction (B)is similarly fractionated by low-melting agarose electrophoresis, and agel band containing the DNA fragment-B of 1200 bp as the amplificationproduct is cut and separated out of the agarose gel. Furthermore, thesetwo cut pieces of gel bands are purified separately by using a DNApurification kit, and thereby individually a purified product of the DNAfragment-D and a purified product of the DNA fragment-B are prepared.

Additionally, there is conducted a second step of PCR method forpreparing a 1200-bp DNA fragment which carries therein a DNA sequence asformed by a modification of the nucleotide sequence of SEQ ID No. 1 ofSequence Listing, with the base cytosine at the position 497 beingreplaced by thymine (namely, said DNA sequence is corresponding to themodified DNA-166A of the nucleotide sequence of SEQ ID No. 7), and which1200-bp DNA fragment further contains an extension part having a KpnIcleavage site at the 5′ terminus of the DNA fragment and also anextension part having a SacI cleavage site at the 3′ terminus thereof.To this end, the purified product of the DNA fragment-D (said sequenceof 480 bp) to be used as the template, and the purified product of theDNA fragment-B (said sequence of 1200 bp), as well as the primer RV-1and the primer FW-1 are added to an amplification mixture for PCRmethod, followed by making subsequent amplification reactions therein.After the termination of the reactions, the amplification reactionsolution obtained is fractionated by low-melting agaroseelectrophoresis. A gel band containing the desired DNA fragment of1200-bp length (referred to as DNA fragment-E) is cut and separated outof the agarose gel.

The gel band so cut out is purified with a DNA purification kit, toproduce a purified product of the DNA fragment-E. This DNA fragment-Ehas a structure such that this DNA fragment carries therein thenucleotide sequence corresponding to the modified DNA-166A sequence ofthe second aspect of the invention and also has an extension part havinga KpnI cleavage site at the 5′ terminus of the DNA fragment and has anextension part having a SacI cleavage site at the 3′ terminus thereof.

The DNA fragment-E is used to produce a DNA fragment carrying thereinthe objective modified DNA-166A sequence (a second example of themodified DNA of the second aspect of the invention). For this purpose,the DNA fragment-E is treated with restriction endonucleases KpnI andSacI. Thereby, from the DNA fragment-E is cut out and separated a DNAfragment which carries therein the modified DNA-166A sequence, whichcontains an extension part having a SacI cleavage site at the 3′terminus of the DNA fragment, and of which the 5′ terminus starts atATG.

In such manner, a sample of the DNA fragment containing the DNA sequencecorresponding to the modified DNA-166A sequence can be prepared. Bysubsequently ligating this DNA sample to the plasmid vector pBluescriptII SK (+) with using a DNA ligation kit in the same way as described inthe item (4) of the Section of explaining “Method for preparing the DNAfragment containing the modified DNA-158N sequence”, there is produced arecombinant vector. Then, Escherichia coli XL1-Blue MRF′ is transformedwith the resulting recombinant vector, followed by progressing theproliferation of the resulting transformed Escherichia coli cell, andthus there can be cloned the plasmid containing the modified DNA-166Asequence.

Then, the thus cloned plasmid containing therein the modified DNA-166Asequence is recovered from the cultured cells of Escherichia coli. Then,the plasmid as recovered is treated in the same way as described in theitem (5) of the Section of explaining “Method for preparing a DNAfragment containing the modified DNA-158N sequence”. Thereby, a DNAfragment of 1143 bp can be prepared in the form of a water-solublepowder and this DNA fragment is a DNA fragment which carries therein theobjective modified DNA-166A sequence.

In the above descriptions, the DNA fragment containing therein themodified DNA-158N sequence as well as the DNA fragment containingtherein the modified DNA-166A sequence according to the second aspect ofthe invention have been prepared by a method of genetic engineeringtechnology. With making reference to the nucleotide sequences describedin SEQ ID No. 5 and No. 7, these two DNA fragments may be prepared alsoby conventionally known chemical synthesis of polynucleotides.

Specific embodiments of the second aspect of the invention are describedin the above for such case where thymine replaces the adenine atposition 473 or cytosine at position 497 of the nucleotide sequence ofthe DNA according to the first aspect of the invention. While, when theDNA of the first aspect of the invention is used as template and when acombination of synthetic oligonucleotides having appropriately preparednucleotide sequences is used as primers, it is possible to prepareanother modified DNA which has such a nucleotide sequence where a basepresent at a position away from the position 473 or 497 of the DNA ofthe first aspect of the invention has been replaced by another base.

Still further, the inventors have now further promoted additionalinvestigations. Consequently, the inventors have now found that thenovel DNA for encoding DHDPS according to the first aspect of theinvention, as well as the novel modified DNA for encoding DHDPSaccording to the second aspect of the invention can be integrated into arecombinant vector and can then be introduced in a plant, so as topermit these DNAs to be expressed in the plant, when use is made of theconventionally known biotechnology manipulation procedures whichcomprise integrating an exogenous gene into a plant to transform theplant, and thereafter allowing the exogenous gene to express in theresulting transgenic plant.

In accordance with a third aspect of the invention, therefore, there isprovided a transgenic plant characterized in that the plant comprisessuch plant cells that have been transformed by the integration of theplant cells with a recombinant vector which carries therein either theDNA for encoding the rice dihydrodipicolinate synthase according to thefirst aspect of the invention, or the DNA for encoding a protein havingthe dihydrodipicolinate synthase activity according to the second aspectof the invention, and wherein the inserted DNA is such one which can beexpressed in the host cells.

The inventors now have additionally found that, when the transgenicplant as transformed by the integration of the DNA of the first orsecond aspect of the invention is such plant which can fructify a seedby cultivation of the plant, the seeds of the plant can be harvested bycultivating the transgenic plant under normal conditions.

In a fourth aspect of the invention, therefore, there is provided theseed of a transgenic plant, characterized in that said seed is recoveredby cultivating the transgenic plant comprising such plant cells whichhave been transformed by the integration of the plant cells with arecombinant vector having carried therein the DNA for encoding the ricedihydrodipicolinate synthase according to the first aspect of theinvention, wherein the inserted DNA can be expressed in the host cells,and then collecting the seed from the plant having fructified duringcultivation of the plant.

In a fifth aspect of the invention, furthermore, there is provided theseed of a transgenic plant, characterized in that said seed is recoveredby cultivating a transgenic plant comprising such plant cells which havebeen transformed by the integration of the plant cells with arecombinant vector having carried therein the DNA of the second aspectinvention for encoding a protein having the dihydrodipicolinate synthaseactivity, wherein the inserted DNA can be expressed in the host cells,and then collecting the seed from the plant having fructified during theplant cultivation of the plant.

In a sixth aspect of the invention, there is provided as a novelmicroorganism, a transformed cell of Escherichia coli, characterized inthat the cell carries therein such a recombinant plasmid which has beenprepared by ligating a DNA fragment containing therein the DNA sequenceshown in SEQ ID No. 1 of Sequence Listing, to the restrictionendonuclease EcoRI cleavage site of the plasmid vector pBluescript IISK(+) with using a DNA ligation kit, and wherein the transformed cell ofEscherichia coli can proliferate stably in the presence of ampicillin.

The transformed cell of Escherichia coli according to the sixth aspectof the invention may be the cell of Escherichia coli DAP8-1 strain whichis deposited as under Accession No. FERM BP-6310 in terms of theBudapest Treaty at the National Institute of Bioscience andHuman-Technology, the Agency of Industrial Science and Technology, inJapan.

Various diverse kinds of plants can be transformed by using, as anexogenous gene, the DNA of the first aspect of the invention or the DNAof the second aspect of the invention. The method for inserting theinventive DNA as the exogenous gene into a plant for planttransformation may be any of various biotechnology methods which areconventionally known for that purpose.

Next, a method, which can be worked suitably for inserting the DNA ofthe first aspect of the invention or the DNA of the second aspect of theinvention as an exogenous gene into the rice plant, is summarilydescribed herein below. This method is illustrated in Example 4hereinafter.

(1) Preparation of a Recombinant Vector for Insertion of Exogenous Gene

When the known plasmid vector pBI221 (manufactured by Clontech, Co.)comprising the 35S promoter of cauliflower mosaic virus and the NOSterminator and an ampicillin-resistant gene is treated with restrictionendonucleases XbaI and SacI in a buffer, there can be produced such avector DNA fragment of about 3.8-kb length which is cut at the XbaIcleavage site located at downstream of the 35S promoter and which is cutat the SacI cleavage site located at upstream of the NOS terminatorpresent in said plasmid vector.

An aqueous solution of said vector DNA fragment of about 3.8 kb is mixedwith an aqueous solution of the DNA of this invention, and the resultingmixture is subjected to a ligation reaction by treating the mixture witha DNA ligation kit. Thereby, there can be produced such a recombinantvector, wherein the DNA of this invention is inserted and ligated at theposition between the 35S promoter region and the NOS terminator regionof said vector DNA fragment.

The recombinant vector so produced is then integrated in Escherichiacoli XL1-Blue MRF′, to prepare the transformant cells of Escherichiacoli.

The resulting transformed cells of Escherichia coli are inoculated toand cultured in a culture medium containing an antibiotic, ampicillin,to afford a number of colonies of Escherichia coli which are resistantto ampicillin. Furthermore, these colonies are separately cultured andproliferated in further volumes of the culture media containingampicillin.

From the ampicillin-resistant Escherichia coli cells as proliferated inthe individual bacteria colonies, then, there are separately harvestedthe plasmids. The so harvested plasmids are composed of various plasmidswherein the DNAs as inserted are linked with each other in variabledirections. The various plasmids as harvested from each bacteria colonyare then digested with restriction endonucleases XbaI and SacI. Theresultant digestion solution containing the various digested DNAfragments is further subjected to agarose gel electrophoresis. By theanalysis of the lengths and the nucleotide sequences of these digestedDNA fragments, it is possible to select an appropriate recombinantplasmid (of a size of about 4.9 kb) wherein the DNA of this inventionhas been inserted and ligated in the normal orientation at thedownstream of the 35S promoter present in the recombinant vector.

The so selected recombinant vector carrying therein the DNA of the firstaspect of the invention, which has been inserted and ligated therein inthe normal orientation, is now referred to as “vector pDAP”. The soselected recombinant vector carrying therein the modified DNA-158Nfragment of the second aspect of the invention, which has been insertedand ligated in the normal orientation therein, is now referred to as“vector p158N”. Additionally, the recombinant vector carrying thereinthe modified DNA-166A fragment of the second aspect of the invention,which has been inserted and ligated therein in the normal orientation,is now referred to as “vector p166A”. All these selected recombinantvectors contain one of the DNA fragments of this invention as well asthe 35S promoter region, the NOS terminator region and theampicillin-resistant gene (Am^(r)).

(2) Preparation of Rice Callus

From the completely ripe rice seeds are removed the outer grain shells.The resulting hulled rice seeds having outer skin are sterilized in anaqueous ethanol solution and then in a dilute aqueous solution of sodiumhypochlorite, and are rinsed with sterile water.

The sterilized and rinsed rice seeds having outer skin are then placedin a callus-forming MS culture medium having been supplemented withsugar, a plant hormone, 2,4-PA and agar. Rice calluses are formed whenthe rice seeds are then cultured at 28° C. under irradiation ofsun-light at 1500 to 2500 lux for 15 to 18 hours per day for consecutive40 to 50 days. The callues are cut out and separated from the albumenpart of each rice seed, and then sieved to afford calluses each of adimension of 1 mm or less.

(3) Preparation of Whiskers for Transfer of Gene

A great number of a commercially available whiskers (microfineneedle-like bodies) made of potassium titanate is placed and sterilizedin ethanol within a small tubular container. The ethanol is thenabsolutely removed from the container through evaporation. Sterile wateris placed in the tubular container containing the sterilized whiskerstherein, in order to rinse the whiskers. The wash liquor is then removedby centrifugation. To the tubular container containing therein therinsed whiskers, there is added a liquid R2 medium, thereby to prepare asuspension of the whiskers.

(4) Preparation of Materials for Introduction of Exogenous or ForeignGene in the Rice Callus Cell

The above-mentioned recombinant vector (namely, the vector pDAP, thevector p158N or the vector p166A) carrying the DNA of this invention asnormally inserted and ligated therein, which has been prepared asdescribed above in the item (1), is dissolved in a TE buffer, thereby toprepare a solution of the recombinant vector.

In the said tubular container containing therein the above whiskerssuspension are then placed and charged said calluses of a dimension of 1mm or less. The charged amount of the whiskers is adjusted to 1 to 100mg per 1 ml of PCV (Packed Cell Volume) of the callus. The resultantmixture present in the container is then agitated and centrifuged. Theresulting supernatant is discarded, and a mixture of the rice calluscells with the whiskers is thus obtained as the precipitate.

To said precipitate which is composed of the mixture of the callus cellsand the whiskers, there are added a solution of the recombinant vectorcarrying the DNA of this invention (namely, the vector pDAP, the vectorp158N or the vector p166A) and a solution of the known plasmid vectorp35SC-SS containing therein a gene resistant to a herbicidephosphinothricin as a selection marker. The resultant admixture is thensufficiently shaked. In this manner, a homogenous mixture which iscomprising the callus cells, the whiskers, the recombinant vectorcarrying the DNA of this invention and the plasmid vector p35SC-SS, canbe prepared. The homogeneous mixture is then repeatedly subjected to aseries of centrifugation and shaking, thereby to prepare the mixturewhich has been made more homogenous.

(5) Manipulation of Exogenous Gene for Insertion of the Gene into RiceCallus

The above-mentioned homogenous mixture comprising the callus cells, thewhiskers, the recombinant vector carrying the DNA of this invention andthe plasmid vector p35SC-SS, which has been prepared as above in theitem (4), is then treated by ultrasonic irradiation. The irradiatingultrasonic wave may be of a frequence of 10 to 60 kHz, and theirradiation intensity may be 0.1 to 1 W/cm². By effecting the ultrasonictreatment of said homogeneous mixture for 30 seconds to 2 minutes, therecombinant vector which is carrying the DNA of this invention and theplasmid vector serving as the selection marker, can be introduced, withaid of the physical ultrasonic action and the whisker's action, into thecallus cells which are to be integrated with the DNA of this invention.

(6) Selection of Callus Cells as Transformed with the IntroducedRecombinant Vector

The mixture which has ultrasonically been treated as described in theabove, is then rinsed with R2 liquid culture medium. The resultingrinsed mixture is centrifuged to isolate the callus cells from thewhiskers. The so isolated callus cells are the transformant cells,wherein the introduced plasmid vector has been carried therein.

The resultant callus cells carrying the introduced plasmid vector areplated on a plant cell-incubating medium which was prepared by addingsucrose and a plant hormone 2,4-PA to the routine R2 medium. The calluscells are incubated at 27 to 29° C. under shaking and irradiation oflight at 1,500 to 2,000 lux for 15 to 20 hours per day. Thereby, thecallus cells can proliferate by occurrence of the cell division.

After incubation for 2-3 day, the resulting suspension of the thusdifferentiated plant cells is spread evenly on a selecting medium whichessentially comprises an N6 medium as supplemented with sucrose, 2,4-PA,Gelrite and a herbicide, phosphinothricin. Then, the plant cells areincubated at 27 to 28° C. under irradiation of light at 1500 to 2000 luxfor 15 to 20 hours per day for consecutive 25 to 30 days. In this way,there can be prepared the phosphinothricin-resistant plant cells whichhas been transformed with the introduced vector.

(7) Re-selection of Transgenic Plant Cells

From among the phosphinothricin-resistant transgenic plant cellsobtained as above, there are re-selected and separated only thetransgenic plant cells containing a sufficient amount of the DNA of thisinvention as the exogenous gene therein.

For this purpose, the above transformed plant cells are transplanted ona re-selecting medium which essentially comprises an N6 medium assupplemented with sucrose, 2,4-PA, Gelrite and a lysine-analog, AEC[namely, S-(2-aminoethyl) cysteine].

The plant cells on the re-selecting medium are cultured at 27 to 28° C.under irradiation of light at 1,800 to 2,000 lux for 15 to 16 hours perday, for 25 to 30 days.

The transgenic plant cells containing the sufficient amount of the DNAof this invention as the exogenous gene is able to grow even in thepresence of AEC having a cell proliferation-inhibiting activity which iscontained in the re-selecting medium, because said transgenic plantcells are resistant to AEC. The AEC-resistant plant cells which couldgrow and were cultured in the above re-selecting medium containing AECadded, are then selected and separated.

(8) Regeneration of a Plant from the AEC-resistant Transgenic PlantCells as Re-selected

The AEC-resistant transgenic plant cells which have thus beenre-selected are then transplanted onto a differentiating medium forregeneration of plant body, which culture medium comprises the MS mediumfor cultivation of plant tissue and has been supplemented with sucrose,benzyladenine and naphthalene acetate, as plant hormone, and Gelrite.

The plant cells as transplanted on the differentiating medium arecultured at 27 to 28° C. under irradiation of light at 1,800 to 2,000lux for 15 to 16 hours per day, for 25 to 30 days. The so culturedtransgenic plant cells have been differentiated, to regenerate the budand root of the plant.

After the plumules having the regenerated bud and root have grown to alength of 10 to 30 mm, the plumules are transplanted onto to anacclimating medium which essentially comprises an MS medium assupplemented with sucrose and Gelrite. The plumules as transplanted areincubated on the acclimating medium at 27 to 28° C. under irradiation oflight at 1,800 to 2,000 lux for 15 to 16 hours per day, for consecutive18 to 20 days.

In this manner, a transgenic plant can be regenerated. The resultingbody of the transgenic plant is transplanted in soil and then cultivatedunder normal conditions in a green house, to allow the plant to grownormally and to fructify rice seed by cultivation for 3 to 6 months.

(9) Verification of the Exogenous Gene as Introduced

Green leaves are harvested from the regenerated body of the transgenicrice plant so obtained. The harvested leaves are frozen in liquidnitrogen and then disrupted. From the disrupted pieces of the leaves isthen extracted the DNA according to the method of J. Sambrook et al.,(described in the Molecular Cloning, 2-nd edition, Cold Spring HarborLaboratory Press, 1989).

Further, an oligonucleotide of the nucleotide sequence of SEQ ID No. 14of Sequence Listing, as well as an oligonucleotide of the nucleotidesequence of SEQ ID No. 15 are chemically synthesized to be used as theprimers.

The DNA as extracted from the regenerated body of the transgenic riceplant is used as the template and the abovementioned two types of thesynthetic oligonucleotides are used as primers, to carry out the PCRmethod, whereby said DNA can be amplified according to the known PCRmethod. The resulting amplification solution is then fractionated byagarose electrophoresis in a conventional manner. Then, there isseparated and collected such a gel band carrying only the DNA fragmentwhich is corresponding to the DNA of said introduced exogenous gene butwhich is presented among the various DNA fragments of the DNA asextracted from said regenerated transgenic rice plant.

By making Southern analysis of the nucleotide sequence of the DNAfragment which is contained in the so collected gel band, it can beidentified whether said DNA fragment is corresponding to the DNA of thisinvention.

In the foregoing descriptions, there has been described a preferablemethod for transforming the rice plant which comprises introducing theDNA of this invention as an exogenous gene into the rice plant. While,the DNA of this invention may also be used for the transformation ofother plant species, by introduction of the DNA of this invention notonly in rice plant but also in other plant species. Accordingly, amethod for introduction of the DNA of this invention into the body of aplant will be described in general hereinbelow.

The plants in which the DNA of this invention may be introduced,include, for example, rice, corn, wheat, barley, and other monocots, aswell as dicots such as tobacco, soy bean, cotton, tomato, Chinesecabbage, cucumber and lettuce, but are not limited thereto. It isadvantageous that cultured cells are at first prepared from one of theseplants, and the DNA of this invention is introduced as an exogenous geneinto the resulting cultured cells.

The preparation of the cultured cells for use in the introduction of theDNA of this invention may be done with employing any of explant asderived from plant, which may be, for example, such explant as derivedfrom scutellum, meristem, pollen, anther, lamina, stem, petiole and rootof the plant.

It is advantageous that the explant as above is placed and incubated ona medium for forming callus, which may be for example such a medium thatis prepared by admixing a plant tissue-incubating medium comprisinginorganic components and vitamins as the essential components, with aplant hormone such as 2,4-PA (2,4-dichlorophehoxyacetic acid) in anamount of 0.1 to 5 mg/liter and carbon sources such as sucrose in anamount of 10 to 60 g/liter and Gelrite in an amount of 1 to 5 g/liter.Said plant tissue-incubating medium may be the MS medium (Murashige etal., Physiological Plantarum, Vol. 15, pp. 473-497, 1962), or the R2medium (Ojima et al., Plant and Cell Physiology, Vol. 14, pp. 1113-1121,1973), or the N6 medium (Chu et al., In Proc. Symp. Plant TissueCulture, Science Press Peking, pp. 43-50, 1978). The resulting cells ascultured in the above manner may then be used for the introduction ofthe DNA of this invention thereinto.

The plant cells into which the DNA of this invention may be introducedare preferably, for example, dedifferentiated cultured cells such ascallus and suspended cells thereof, or cultured cells made ofadventitious embryo and shoot primordium, or callus cells or suspendedcells thereof as prepared from the cells of plant tissues such as leaf,root, stem, embryo and meristem of the plant.

To prepare the cultured cells for use in the introduction of the DNA ofthis invention thereinto an explant piece may be placed and incubated ona callus-forming medium. In this case, the incubation period which isrequired for the formation of the cultured cells for use in theintroduction of the DNA of this invention into said cultured cells isnot specifically limited. However, it is essential to regenerate theresulting transgenic plant, and hence it is important to employ suchcultured cells which are capable of producing the regenerated plant fromthe cultured cells, and which are namely cultured cells as harvestedwithin the period of time during which the cultured plant cells canstill retain their ability to regenerate the plant therefrom.

The cultured cells for use in the introduction of the DNA of thisinvention therein may be in the form of any suspended cells which areobtained by culturing the cells in a liquid medium, so long as thecultured cells as suspended are retaining the ability to regenerate theplant.

In order to introduce the inventive DNA (namely, the DNA of thisinvention) in a plant cell, it is at first necessary to prepare such arecombinant vector in which the inventive DNA has been inserted in anexpressing vector. The recombinant vector so prepared is necessary to besuch a recombinant vector which is so constructed and arranged that theinventive DNA is located at downstream of the expressing promotor in therecombinant vector and a terminator is located downstream of theinventive DNA, whereby the inserted DNA can be made to express in thetransformed plant after the introduction of the DNA into the plant.Depending on the type of a method as employed for the introduction ofDNA in plants, the recombinant vector which is to be used for thatpurpose may be any of the various vectors which have usually beenutilized in routine transformation of plants. For example, it isconvenient to employ such plasmid vectors which are capable ofproliferating in the cell of Escherichia coli, such as ones of pUCseries and pBR322 series in cases when there are used direct methods forthe introduction of DNA in plant cells, which are operable byelectroporation process, particle gun process and whisker process.While, it is convenient to employ such plasmid vectors such as one ofplan series is a case when there is used the Agrobacterium process.

The promoters which is to be located at upstream of the inventive DNAwithin in said recombinant vector include, for example, CaMV35S asderived from cauliflower mosaic virus [see The EMBO Journal, Vol. 6, pp.3901-3907, 1987; or JP-A-6-315381]; ubiquitin promoter as derived fromcorn [JP-A-2-79983]; and phaseolin promoter [see Plant Cell, Vol. 1, pp.839-853, 1989]. Further, the terminator which is to be located atdownstream of the inventive DNA within the said recombinant vector maybe, for example, the terminator as derived from cauliflower mosaicvirus, or the terminator as derived from nopaline synthase gene [see TheEMBO Journal, Vol. 6, pp. 3901-3907, 1987]. However, any promoter orterminator which is capable of functioning in plants may satisfactorilybe used.

In order to enable it to efficiently select the plant cells which havebeen transformed by the introduction of the inventive DNA therein, it ispreferable that the recombinant vector for use therefor is introducedinto the plant cells, along with a plasmid vector which is containing agene useful as an appropriate selection marker. For this purpose, as theselection marker gene is used either a hygromycin phosphotransferasegene which is resistant to an antibiotic hygromycin, or a neomycinphosphotransferase gene which is resistant to kanamycin and gentamycin,or an acetyltransferase gene which resistant to a herbicide,phosphinothricin [see The EMBO Journal, Vol. 6, pp. 2513-2518, 1987; orJP-A-2-171188].

The methods which may be used for the introduction of the inventive DNAas the exogenous gene into a plant, may include the Agrobacteriumprocess [Bio/technology, Vol. 6, pp. 915-922, 1988], electroporationprocess [Plant Cell Rep., Vol. 10, pp. 106-110, 1991]; particle gunprocess [Theor. Appl. Genet., Vol. 79, pp. 337-341, 1990]; and whiskermethod, but to which this invention is not limited.

According to the whisker method for the introduction of the inventiveDNA as the exogenous gene into the plant, the whiskers suitably usablemay specifically be of a diameter of 0.01 to 10 μm, preferably 0.5 to 1μm and of a length of 1 to 100 μm, preferably 3 to 40 μm. The whiskersmay be made of a material such as potassium titanate, calcium carbonate,aluminium borate, silicate nitride, zinc oxide, basic magnesium sulfate,magnesia, magnesium borate, carbon graphite, calcium sulfate, sapphire,and silicon carbide. The whisker is preferably made of potassiumtitanate, calcium carbonate, or aluminium borate.

The whisker having received no surface treatment can here be used assuch. However, the rate of the transformation of plant cells can beraised when use is made of a whisker having been treated so as to conferbasic functional groups on the surface of the whisker, or preferably awhisker having the surface treated with a surface-treating agent.

Any of a compound capable of forming a covalent bond with the whiskersurface may satisfactorily be used as the treating compound which canconfer the basic functional groups to the whisker surface. The compoundusable for this purpose is preferably a silane coupling agent, morepreferably a silane coupling agent having a basic functional group. Asthe silane coupling agent, use can be made of a basic, silane couplingagent such as 3-(2-aminoethoxyaminopropyl)-trimethoxysilane and3-aminopropyl-triethoxysilane. Any silane coupling agent having thebasic functional group can satisfactorily be used.

An appropriately adjusted quantity of the plant cells is mixed with thewhiskers at first in a liquid medium. The volume of the plant cells tobe mixed with the whiskers is not limited to a specific value. Thevolume of the whiskers to be mixed into a liquid medium, along with theplant cells, may be adjusted, depending on the volume of the plantcells. Per 1 ml of PCV for the plant cells, the whiskers may be added inan amount of 1 to 100 mg, preferably 4 to 40 mg.

In this way, a mixture of the plant cells, the whiskers and therecombinant vector carrying therein the inventive DNA as dispersed inthe liquid medium is prepared. The mixture is then centrifuged. Forexample, said mixture may be centrifuged at a centrifugal acceleratedrate of 3,000 to 50,000×g, preferably 10,000 to 30,000×g for 10 secondsto 40 minutes, preferably 5 to 10 minutes. Thereafter, the resultingprecipitate is subjected to the ultrasonic treatment. For example, theultrasonic treatment may comprise the irradiation of ultrasonic wave ata frequency of 1 k to 1 MHz, prefer-ably 10 to 60 kHz and at anintensity of 0.01 to 10 W/cm² ₁ preferably 0.1 to W/cm² for anirradiation period of 0.2 second to 20 minutes, preferably 30 seconds to2 minutes.

From the mixture having received the ultrasonic treatment, there areseparated the plant cells by centrifugation. The so harvested plantcells contain the recombinant vector carrying therein the insertedinventive DNA, as well as the plasmid vector usable as the selectionmarker.

The thus harvested plant cells which are carrying the exogenous gene asintroduced therein, are then rinsed in a liquid medium. Thereafter, theplant cells are plated and cultured in a known selecting medium whichcontains an appropriate, selecting chemical agent varying depending onthe type of the selection marker gene as integrated in the plant cells.Thereby, the cultured transgenic plant cells can be prepared.

In order to re-select the transformed plant cells which contain therecombinant vector carrying the inventive DNA therein in a sufficientlyeffective amount of the vector, said transformed plant cells are thencultured in a re-selecting medium containing lysine analog added as acell proliferation-inhibiting agent. As the lysine-analog,S-(2-aminoethyl)-cysteine (AEC) or O-(2-aminoethyl)-serine, for example,may be added at a concentration of 10 mg/liter to 1000 mg/liter,preferably 100 mg/liter to 300 mg/liter to the re-selecting medium.

The transformed plant cells which are carrying the recombinant vectorcontaining the inserted inventive DNA and are carrying the vector as theselection marker, may be re-selected in this manner, and are thenincubated to regenerate the plant therefrom. The plant can beregenerated according to a known technique. For example, the plant canbe regenerated by plating and incubating the above, re-selectedtransformed plant cells in a known medium which is conventional for theplant regeneration.

The above, re-selected transformed plant cells are thus plated in themedium for the plant regeneration and may then be incubated therein at atemperature of 15 to 30° C., preferably 20 to 28° C., under irradiationof light at 500 to 2,000 lux, preferably 800 to 1,000 lux for anincubating period of 20 to 60 days, preferably 30 to 40 days.

Thereby, from the so incubated individual plant cells can be regeneratedthe bodies of the transformed plant in which the recombinant vectorcarrying therein the exogenous gene containing the inventive DNA hasbeen introduced.

The resulting plant as regenerated from said transformed plant cells isthen cultivated in an acclimation medium. Thereafter, the so acclimated,regenerated plant is cultivated under normal cultivation conditions in agreenhouse. By the cultivation of the plant for 3 to 6 months, the plantreaches its mature stage to fructify seeds, which can be harvested.

The presence of said exogenous gene which is introduced in thetransgenic plant as regenerated and cultivated in the above manner, canbe confirmed by effecting the analysis of the nucleotide sequence of theDNA present in said plant according to known PCR method and Southernmethod [Southern, J. Mo. Biol., Vol. 98, pp. 503-517, 1975].

In this case, extraction of the DNA from said transqenic plant may bepracticed according to the known method of J. Sambrook [MolecularCloning, 2-nd edition, Cold Spring Harbor Laboratory Press, 1989].

When there is conducted by PCR method the analysis of the inventive DNApresent as the exogenous gene in the regenerated transgenic plant, theDNA as extracted from the regenerated transgenic plant is used astemplate, and further, synthetic oligonucleotides having the nucleotidesequences as appropriately chosen depending on the DNA of the firstaspect of the invention or the modified DNA of the second aspect of theinvention are used as primers, and then they together are added to areaction mixture for PCR method to carry out the amplification reactionsof DNA. The amplification reactions for this purpose comprise repeatingthe modification of the DNA and the annealing and extension of DNA byseveral tens of times, so that an amplified product of the DNA fragmentcontaining the DNA sequence of this invention can be produced.

The resultant amplification reaction solution containing said amplifiedproduct may then be subjected, for example, agarose gel electrophoresisin order to fractionate the various amplified DNA fragments so produced.An agarose gel band which carries the DNA fragment containing thereinthe DNA sequence corresponding to the DNA of this invention as theintegrated exogenous gene is then cut out and separated from the agarosegel. By making Southern analysis of the nucleotide sequence, it can bedetermined whether or not the nucleotide sequence of the DNA fragmentcontained in the gel band thus cut out and separated is corresponding tothe DNA of this invention.

In a seventh aspect of the invention, there is further provided arecombinant vector as prepared by insertion of a DNA fragment carryingtherein the DNA sequence of 1143 bp shown in SEQ ID No. 1 or No. 5 orNo. 7 of Sequence Listing, into such a plasmid vector which comprises acauliflower mosaic virus-derived promotor capable of expressing in aplant, as well as the NOS terminator and an ampicillin-resistant gene,and wherein, in the recombinant vector, the inserted DNA sequence of1143 bp is located so as to be controllable by said promoter.

The plasmid vector mentioned just above is preferably the aforesaidplasmid vector pBI221.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows diagrammatically a flow chart of depicting the proceduresfor preparing from the vector pBI221, the aforesaid vector p158N whichis a recombinant vector useful for the introduction of an exogenous geneto be used for the transformation of cultured cells of rice in Example 4hereinafter, and which carries therein the modified DNA-158N sequenceaccording to the second aspect of this invention.

FIG. 2 depicts diagrammatically the structure of the vector p35SC-SSwhich contains the phosphinothricin gene SS as the selection marker andwhich may be introduced, along with the vector p158N, into the culturedcells of rice, in Example 4 hereinafter.

PREFERRED EMBODIMENTS OF THE INVENTION

Next, the first aspect of this invention is illustrated with referenceto Example 1 which illustrates the preparation of a DNA fragmentcarrying the rein the DNA sequence of 1143 bp according to the firstaspect of the invention (that is, the DNA sequence of the nucleotidesequence shown in SEQ ID No. 1 of Sequence Listing, namely theDHDPS-DNA-1143 sequence). Further, the second aspect of the inventionwill be illustrated with reference to Example 2 which illustrates thepreparation of a DNA fragment carrying therein the modified DNA-158Nsequence of 1143 bp according to the second aspect of the invention(that is, the DNA sequence of the nucleotide sequence shown in of SEQ IDNo. 5 of Sequence Listing), as well as and with reference to Example 3which illustrates the preparation of a DNA fragment carrying therein themodified DNA-166A sequence of 1143 bp according to the second aspect ofthe invention (that is, the DNA sequence of the nucleotide sequenceshown in SEQ ID No. 7 of Sequence Listing).

Furthermore, the third aspect of this invention is illustrated withreference to Example 4 which illustrates the method for transforming arice plant by introduction of the DNA of this invention as inserted inthe recombinant vector, as an exogenous gene, into the rice plant.

The procedures for the experimental manipulation as described in thefollowing Examples are those which are carried out according to themethods described in the Molecular Cloning, 2-nd edition, J. Sambrook etal., Cold Spring Harbor Laboratory Press (1989), unless otherwisestated.

EXAMPLE 1

(1) Preparation of Rice mRNA

Seeds of rice plant (variety: Nippon-bare) were sown. On day 7 of thecultivation, 2 g of the stems and leaves of the young rice plants wasfrozen in liquid nitrogen. The frozen stems and leaves were disrupted ina mortar. From the disrupted plant material so obtained was extracted atotal RNA of about 2 mg, according to the known AGPC method (the methodusing acid guanidinium thiocyanate phenol-chloroform) (ExperimentalMedicine, Vol. 9, No. 15 (November issue), pp. 99-102, 1991). Then, mRNAwas isolated from the resulting total RNA by means of an mRNApurification kit (mRNA Purification Kit; manufactured by PharmaciaBiotech, Co., Ltd.). In this manner, the mRNA of rice was obtained at ayield of about 30 μg.

(2) Construction of Rice cDNA Library

From the mRNA as obtained in (1) above were produced cDNAs by means of acDNA synthesis kit (Time Saver cDNA Synthesis Kit; manufactured byPharmacia Biotech, Co. Ltd.).

The resultant cDNAs were linked to a phage vector λgt11, of which theEcoRI cleavage terminus had been treated with the calf intestine-derivedalkaline phosphatase [see DNA Cloning Techniques, IRL Press, Oxford,Vol. 49, 1985; Lamda gt11/EcoRI/ClAP-treated Vector Kit; manufactured bySTRATAGENE CO.). Then, the resulting recombinant vectors were packagedin the λ phage by using an in vitro packaging kit [Gigapack II GoldPackaging Extract; manufactured by STRATAGENE LTD.].

Escherichia coli Y1088 was then infected with the thus producedrecombinant phage and then proliferated. A great number of saidrecombinant phages was produced as plaques of the lysogenized cells ofEscherichia coli. The recombinant phages present in the plaquescomprised a variety of the recombinant phages containing therein theinserted cDNAs derived from rice, and these recombinant phages were usedas a rice cDNA library.

(3) Construction of Primers for PCR Method

For the purpose of preparing such a DNA probe for PCR method, whichserves for cloning of the cDNA fragment to encode the rice DHDPS, aprimer was designed at first. For this purpose, and also with referenceto the known nucleotide sequences of the DHDPS genes of wheat and cornand with reference to the known DHDPS amino acid sequences of DHDPS, thefollowing two types of oligonucleotides were prepared as primers No. 1and No. 2.

Primer No. 1 (having the nucleotide sequence of SEQ ID No. 3 of SequenceListing):

5′-GTAATAGTTGGAGGAACAACAGGAG-3′

Primer No. 2 (having the nucleotide sequence of SEQ ID No. 4 of SequenceListing):

5′-GAGCTGAGCCAGAGCAGTGTTGAG-3′

The aforementioned two oligonucleotides were synthetized by using a DNAsynthesizer (Model 391; manufactured by Applied Biosystems, Co. Ltd.)and purifying with ion exchange HPLC.

(4) Preparation of Probe DNA

The above-mentioned two types of the oligonucleotides were used in anamount of 10 p.M each as the first primer and second primer,respectively. The rice cDNA library comprising the recombinant phageswhich were produced above in (2), was used as the template. Theseprimers and template together were added to 50 μl of an amplificationmixture for PCR [comprising 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl₂, 50 mMKCl, 0.001% gelatin, pH 8.3, and a mixture of 4 dNTPs each at 2.5 mM,and a DNA polymerase TaKaRa Ex Taq of 2.5 units], to effectamplification of DNA. The amplification mixture here used had beenprepared by using a PCR kit (PCR Amplification Kit; manufactured byTaKaRa Brewery Co., Ltd.).

The amplification reaction of the DNA by PCR method was effected byrepeating 35 times a reaction cycle which consisted of three reactions,namely denaturation at 94° C. for 30 seconds, annealing at 55° C. forone minute, and extension at 72° C. for 2 minutes, with using a pCRreaction apparatus (DNA Termal Cycler 480, manufactured by PERKINELMER).

Thus, the amplification products of the DNA fragments which constituteparts of the DNA sequence corresponding to the rice DHDPS gene wereproduced. And, these DNA fragments were then collected as the probeDNAs. These probe DNAs were used for the following procedure of cloningthe cDNA library.

(5) Selection of DNA of the Rice DHDPS Gene from the Rice cDNA Library

From the recombinant phages present in the rice cDNA library as preparedin (2) above, there were selected by screening the recombinant phareshaving contained the inserted DNA sequence corresponding to the riceDHDPS gene, with the selection being made by using the probe DNAS asprepared above in (3).

For that purpose, the aforesaid plaques comprising the recombinantphages which are the rice cDNA library as produced above in (2), wereprovided by being formed on a 1.5% agar medium. These plaques on theagar medium were then transferred onto a nylon membrane, High BondN(manufactured by Amersham, Co.). The phage DNAs which were contained inthe phage plaques as transferred onto the nylon membrane, were treatedwith an alkaline denaturation mixture (comprising 1.5 M NaCl, 2.0 MNaOH) for 10 minutes, and with a neutralization mixture (comprising 1.0M Tris-HCl, pH 5, 2.0 M NaCl) for 10 minutes, followed by treating withUV irradiation, so as to immobilize the DNAs on the nylon membrane.

Next, the probe DNAs so produced above in (4) were labeled withdigoxigenin (DIG), to prepare a labeled probe DNAs. The labeled probeDNAs were then plaque-hybridized to said nylon membrane having saidphage DNAs immobilized thereon. The labeled probe DNAs were prepared byusing DIG-ELISA DNA Labeling & Detection Kit (manufactured by BoehringerMannheim, CO.).

In the above case, said nylon membrane having the phage DNAs immobilizedthereon was immersed in a hybridization mixture (comprising 500 mM Na-Pibuffer, pH 7.2, 7% SDS, 1 mM EDTA) at 65° C. for 10 minutes. Then, saidlabeled probe DNAs of 10 ng/ml were added to the hybridization mixturecontaining the nylon membrane therein, followed by effecting thehybridization reaction at 65° C. for 15 hours.

After the termination of the hybridization reaction, the nylon membranehaving the resulting hybridization products was rinsed with a rinsingsolution (comprising 40 mM Na-Pi buffer, pH 7.2, 1% SDS) for 20 minutes.The rinsing procedure was repeated three times. Thereafter, theDIG-ELISA Labeling & Detection Kit was used to detect the desiredrecombinant phage. Four plaques of such recombinant phages which emittedintense signals on an X-ray film due to having received thehybridization, and which are the recombinant phages presumably carryingtherein the integrated DHDPS gene, could thus be detected in among the300,000 phage plaques. Then, the above, four plaques of the recombinantphages could be selected and isolated.

From each of these four recombinant phage plaques so selected, there wasisolated the λ DNA by means of a λ DNA isolation kit (λ DNA PurificationKit; manufactured by STRATAGENE, LTD.).

The above isolation of λ DNA was made by the following procedures. Thus,20 mg/ml DNase I (50 μl) and 2 mg/ml RNase A (200 μl) were added to aliquid culture in which a vast amount of each recombinant phage asselected had been proliferated. The resulting mixture was left to standat ambient temperature for 15 minutes. The resulting culture brothcontaining the proliferated phages was centrifuged at 15,000 rpm at 4°C. for 10 minutes. To the resulting supernatant was added 25 ml of 80%DEAE-cellulose, followed by incubation at ambient temperature for 10minutes. The resulting incubated mixture was centrifuged, and theresulting supernatant were added with 2 ml of 0.5 M EDTA and 770 μl of50 mg/ml Pronase. The resulting reaction mixture was left to stand at37° C. for 15 minutes, followed by being added with 1.5 ml of a 5% CTABsolution [comprising 1% CTAB (cetyl trimethyl ammonium bromide), 50 mMTris-HCl, pH 8.0, and 10 mM EDTA]. The resulting mixture was treated byallowing it to stand at 65° C. for 3 minutes and was then left to standin ice bath for 5 minutes. The respective reaction solutions so preparedwere added with a 1/10-fold volume of 3 M sodium acetate and a 2-foldvolume of ethanol, and the resulting mixture was left to stand at −20°C. for about 6 hours. Subsequently, the resultant solution wascentrifuged, and the precipitated phage DNA was once dried and was thendissolved in 5 ml of water to be stored.

In this way, each of the 4 types of the phage DNAs was isolated. 5 μl ofeach phage DNA was then digested with 10 units of a restrictionendonuclease EcoRI in an H buffer, followed by making the analysis ofthe resultant digestion mixture. Consequently, it was confirmed that allof the above 4 tyes of the isolated phage DNAs were of one and same DNAsequence.

Accordingly, the DNA fragment which is inserted in the said recombinantphage and is judged to carry the DNA sequence of the rice DHDPS gene,could thus be produced by digesting the phage DNA of the recombinantphage as selected and recovered as above, with the restrictionendonuclease EcoRI.

(6) Cloning of cDNA Carrying therein the DNA Sequence Corresponding tothe Rice DHDPS Gene

The DNA fragment, which was produced as above by digesting from the saidrecombinant phage, and which was judged to carry the DNA sequence of therice DHDPS gene, was further inserted in and ligated to the EcoRIcleavage site of a plasmid vector pBluescript II SK(+), by using a DNAligation kit, so that a recombinant plasmid vector was constructed.Escherichia coli XL1-Blue MRF′ was transformed then by inserting the soconstructed recombinant plasmid vector therein.

The insertion of the so constructed recombinant plasmid vector inEscherichia coli XL1-Blue MRF′ as described above was conducted by thefollowing procedures. Thus, 10 μl of the recombinant phage DNA producedin the above was digested with 10 units of a restriction endonucleaseEcoRI in an H buffer, to prepare a digestion mixture. Separately, 10 μlof the plasmid vector pBluescript II SK (+) was similarly digested withEcoRI, to prepare a digestion mixture. Each of the two digestionmixtures so prepared were added with a 1/10-fold volume of 3 M sodiumacetate and a 2-fold volume of ethanol. Each admixture so obtained wasleft to stand at −20° C. for about 6 hours, and the resulting individualDNA solutions were each centrifuged, to separate and collect theprecipitated DNA, which was then dried and dissolved in 5 μl of water.Thus, the aqueous solution of the DNA as derived from the saidrecombinant phage was obtained, and the aqueous solution of the DNA asderived from the plasmid was obtained. These two aqueous DNA solutionswere mixed at each volume of 5 μl together. The resulting mixture wastreated with a DNA ligation kit (manufactured by TaKaRa Brewery Co.,Ltd.), to ligate the above-mentioned two types of the DNAs with eachother. The resulting reaction mixture coming from the DNA-ligationreaction was added with a 1/10-fold volume of 3 M sodium acetate and a2-fold volume of ethanol. The resulting mixture was left to stand at−20° C. for about 6 hours. The resulting mixture was then centrifuged toprecipitate the DNA, which was then separated and dried. The ligatedvector DNA so obtained was dissolved in 10 μl of water.

The resultant aqueous solution (10 μl) of the ligated vector DNA (10 ngDNA), as well as commercially available Escherichia coli XL1-Blue MRF′competent cells (100 μl; manufactured by STRATAGENE, LTD.) were togetherplaced in a 1.5-ml tube, where the resulting mixture was then incubatedfor 30 minutes under ice-cooling, then at 42° C. for 30 seconds, andagain on ice bath for 2 minutes under ice-cooling. Subsequently, the soincubated mixture was added with 900 μl of an SOC liquid medium(containing 2% Bacto-Tryptone, 0.5% Bacto-yeast extract, 10 mM NaCl, 2.5mM KCl, 10 mM MgSO₄, 10 mM MgCl₂, and 20 mM glucose), followed byculturing the Escherichia coli at 37° C. for one hour under shaking.

100 μl of the resulting Escherichia coli culture was plated on an LBagar medium (containing 1% Bacto-Tryptone, 0.5% Bacto-yeast extract,0.5% NaCl, 0.1% Glucose, pH 7.5, 1.5% agar) as supplemented with 50 mg/lof ampicillin, 20 mg/l of X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside), and 20 mg/l of IPTG(isopropyl-β-D-thiogalactopyranoside). The Escherichia coli wasincubated therein at 37° C. for 16 hours, and then such Escherichia colicolonies as colored white were selected as the Escherichia coli whichwas transformed with the said ligated vector DNA. The white coloredcolonies were isolated from the Escherichia coli colonies which werenever colored white.

The so isolated 10 colonies of Escherichia coli which are colored whiteand resistant to ampicillin, were proliferated in a liquid mediumcontaining 50 mg/l ampicillin. From the so further proliferated bacteriaof Escherichia coli was isolated the plasmid, which was then purified byusing a plasmid purification kit (QIA filter plasmid Midi Kit,manufactured by QIAGEN, Co.). By such purification, the plasmid of 50 μg(50 μl) was produced from the transformed Escherichia coli cells presentin the above ampicillin-resistant bacteria colonies.

The resultant plasmid coloned and produced as above is the desiredrecombinant plasmid which is a DNA fragment carrying therein the DNAsequence of the rice DHDPS gene and which is of a size of about 4.3 kb.

(7) Analysis of Sequence of the so Cloned DNA

The whole nucleotide sequence of the DNA fragment of the recombinantplasmid (of a size of about 4.3 kb) obtained as the above mentionedcloned DNA fragment can be determined, by treating the recombinantplasmid with a commercially available nucleotide sequencing kit. Thenucleotide sequence of the DNA which is corresponding to the rice DHDPSgene and is inserted in the DNA fragment of the aforesaid colonedrecombinant plasmid, can be determined, too. The so determinednucleotide sequence of the DNA for encoding the rice DHDPS gene isdescribed in SEQ ID No. 1 of Sequence Listing hereinafter.

Upon making the above determination of the nucleotide sequence of theDNA, the nucleotide sequence of the aforesaid DNA fragment-a wasdetermined by effecting at first a denaturation process by using asequencing kit (Autoread Sequencing Kit; manufactured by PharmaciaBiotech, Co., Ltd.) and then by effecting the sequencing process with anautomatic DNA sequencer [ALF DNA Sequencer II; manufactured by PharmaciaBiotec., Co., Ltd.].

The nucleotide sequence of the DNA which is inserted in the DNAfragment-α as obtained in Example 1 according to this invention andwhich is judged to encode the rice DHDPS gene, consists of 1140 basepairs present in the single open reading frame set out in the SEQ ID No.1 of Sequence Listing. The DNA of the nucleotide sequence as shown inSEQ ID No. 1, which is provided in accordance with the first aspect ofthe invention, encodes a protein consisting of 380 amino acids shown inSEQ ID No. 2 of Sequence Listing. When the amino acid sequence of SEQ IDNo. 2 is compared with the amino acid sequence of wheat DHDPS(JP-A-3-127984) and with the amino acid sequence of corn DHDPS(Molecular & General Genetics, Vol. 228, pp. 287-293, 1991), thesequence of SEQ ID No. 2 shows a 82% homology and 79% homology,respectively, to the latter amino acid sequences. Outside the homologousregion, the DNA sequence of the first aspect of this invention isobviously different from the known DNA sequences of the DHDPS of wheatand corn. The DNA of the first aspect of the invention therefore hassuch a DNA sequence which is specific to rice.

EXAMPLE 2

This Example illustrates the preparation of a DNA fragment which carriedtherein the DNA sequence (namely, a DNA sequence of the nucleotidesequence shown in SEQ ID No. 5 of Sequence Listing) that is designatedas the modified DNA-158N sequence and is produced in accordance with thesecond aspect of the invention.

(1) Construction of Synthetic Oligonucleotide as Primer for PCR Method

A DNA synthesizer (Model-391; manufactured by Applied BioSystems, Co.)was used to construct by chemical synthesis, primer No. 3 which is theoligonucleotide having the nucleotide sequence of SEQ ID No. 9 ofSequence Listing; primer FW-1 having the nucleotide sequence of SEQ IDNo. 11; primer RV-1 having the nucleotide sequence of SEQ ID No. 12; andprimer BS KPN-1 having the nucleotide sequence of SEQ ID No. 13 ofSequence Listing.

Furthermore, a primer having the nucleotide sequence of SEQ ID No. 14and a primer having the nucleotide sequence of SEQ ID No. 15 were alsosynthesized.

(2) Preparation of Template for PCR Method

The DNA fragment as produced by cleavage of the recombinant phage in theitem (6) of Example 1 hereinbefore was such the DNA fragment which wasjudged to carry therein the DNA sequence corresponding to the rice DHDPSgene. Said DNA fragment was now ligated to the EcoRI cleavage site ofthe plasmid vector pBluescript II SK(+) by means of a DNA ligation kit,to construct a recombinant plasmid vector.

The resulting recombinant plasmid vector pDAP8-1 did contain said DNAfragment inserted therein. In order to render that the sites cleavableby restriction endonucleases XbaI and SacI and usable for PCR areconferred to the said DNA fragment as inserted in the recombinant vectorpDAP8-1, the following reactions were carried out.

5 μl of the vector pDAP8-1, as well as 1 μM each of the primer of SEQ IDNo. 14 and the primer of SEQ ID No. 15 were added to 100 μl of anamplification mixture [containing 10 mM Tris-HCl, pH 8.3, 1 mM MgCl₂, 50mM KCl, a mixture of 0.2 mM each of 4 dNTPs, and 2.5 units of LA. TaqDNA polymerase], followed by effecting the amplification reaction. Theamplification reaction was promoted by repeating 30 times such reactioncycle which consisted of three reaction procedures comprisingdenaturation at 94° C. for one minute, annealing at 60° C. for 30seconds, and extension at 72° C. for one minute.

After the termination of the amplification reaction, the amplificationreaction solution so obtained was fractionated by low-melting agaroseelectrophoresis. As the desired, amplified DNA product, there was cutoff and separated a band of about 1160 bp, out of the agarose gel. Fromthe so separated band, was produced a purified DNA fragment(XbaI-DNA-SacI)(DNA fragment-XS) which has an XbaI-cleavage site at the5′ terminus and a SacI-cleavage site at the 3′ terminus of the DNAsequence for encoding the DHDPS gene, by means of Geneclean II Kit(manufactured by Funakoshi, Co., Ltd.).

Further, 10 μl of a commercially available plasmid vector pUC19 wasdigested with 10 units of endonuclease XbaI and 10 units of endonucleaseSacI in an M buffer.

By using the DNA ligation kit, said DNA fragment (XbaI-DNA-SacI) and theresulting cleaved linear product of the plasmid vector pUC19 wereligated together, so that a cyclic recombinant plasmid vector pDAP8-1-XS(of about 3.9 kb) was prepared.

The plasmid vector pDAP8-1-XS so prepared was then used as the templatefor the following PCR method.

(3) Amplification by PCR and Recovery of the Required DNA Fragment

(i) First Step of PCR Method

In order to produce the required DNA fragment by PCR amplification, thefollowing reaction (A) and reaction (B) were conducted as the first stepof PCR method.

More specifically, the reaction (A) comprised adding 5 μl of therecombinant plasmid vector pDAP8-1-XS as the template, as well as 1 μMof the primer FW-1, and 1 μM of the primer No. 3 of SEQ ID No. 9 to 100μl of an amplification mixture [containing 10 mM Tris-HCl, pH 8.3, 1 mMMgCl₂, 50 mM KCl, a mixture of 0.2 mM each of 4 dNTPs, and 2.5 units ofLA Taq DNA polymerase], followed by effecting the amplificationreaction. The amplified DNA product as obtained from the reaction (A) isnow referred to as DNA fragment-A.

Furthermore, the reaction (B) comprised adding 5 μl of the recombinantplasmid vector pDAP8-1-XS as the template, as well as 1 μM of the primerRV-1, and 1 μM of the BS KPN-1 to the amplification mixture of the sameformulation as in the reaction (A), followed by effecting theamplification. The amplified DNA product as obtained from the reaction(B) is now referred to as DNA fragment-B.

The amplification reaction was made by repeating 30 times a reactioncycle which consisted of three reaction procedures comprisingdenaturation at 94° C. for 30 seconds, annealing at 55° C. for 2minutes, and extension at 72° C. for 2 minutes in a PCR reactor [ProgramTemp Control System PC-700; manufactured by Asthec, Co. Ltd.].

After the termination of the amplification reaction, the amplificationreaction mixture coming from the reaction (A) was fractionated bylow-melting agarose electrophoresis. Then, a band containing the DNAfragment-A of 480 bp as the amplified DNA product was cut out of theagarose gel. Furthermore, the amplification reaction mixture coming fromthe reaction (B) was fractionated by low-melting agaroseelectrophoresis. Then, a band containing the DNA fragment-B of 1200 bpas the amplified DNA product was cut out of the agarose gel.

From these gel bands so cut out, there were respectively separated andproduced a purified product of the DNA fragment-A and a purified productof the DNA fragment-B, by using Geneclean II Kit (manufactured byFunakoshi Co., Ltd.).

(ii) Second Step of PCR Method

The second step of the PCR method comprised adding 1 μl of the primerRV-1 and 1 μl of the primer FW-1, and 1 μl each of the DNA fragment-Aand DNA fragment-B as amplified individually by the above reactions (A)and (B), respectively, to 100 μl of an amplification mixture [containing10 mM Tris-HCl, pH 8.3, 1 μM MgCl₂, 50 mM KCl, a mixture of 0.2 mM eachof 4 dNTPs, and 2.5 units of LA Taq DNA polymerase], followed byeffecting the amplification reaction. The amplification reaction wasthen effected by repeating 20 times a reaction cycle which consisted ofthree reaction procedures, namely denaturation at 94° C. for 30 seconds,annealing at 55° C. for 2 minutes, and extension at 72° C. for 2minutes.

After the termination of the amplification reaction, the amplificationreaction mixture was fractionated by low-melting agaroseelectrophoresis. Then, a band containing the desired DNA fragment-C of1200 bp as the amplified DNA product was cut out of the agarose gel.From the gel band so cut out, there was produced a purified product ofthe DNA fragment-C, by using Geneclean II Kit (manufactured by FunakoshiCo., Ltd.).

The DNA fragment-C obtained as above is a DNA fragment of about 1350 bp,which carries therein the DNA-158N sequence (of a length of 1143 bp)having the nucleotide sequence of SEQ ID No. 5 of Sequence Listing, andin which a KpnI cleavage site is located at downstream of the 5′terminus of the DNA-158N sequence and an SacI cleavage site is locatedat upstream of the 3′ terminus thereof.

(4) Cloning of DNA Fragment Containing the Modified DNA-158N Sequence

The DNA fragment-C thus produced was then used to prepare a wellquantity of a DNA fragment containing the target modified DNA-158Nsequence. To this end, the cloning was done according to the followingprocedures.

(i) 10 μg of the DNA fragment-C produced as above was first digestedwith 10 units each of restriction endonucleases KpnI and SacI in an Lbuffer (manufactured by TaKaRa Brewery Co., Ltd.). The DNA fragment asproduce by this digestion is now referred to as DNA fragment-β. Further,10 μg of the pBluescript II SK(+) was digested with 10 units each ofKpnI and SacI in an L buffer (manufactured by TaKaRa Brewery Co., Ltd.),to prepare a truncated plasmid. The resultant digestion solutioncontaining the DNA fragment-β, and the resultant digestion solutioncontaining said truncated plasmid were respectively added with a1/10-fold volume of 3 M sodium acetate and a 2-fold volume of ethanol.The resulting mixtures were individually left to stand at −20° C. forabout 6 hours. Thereafter, the resulting individual solutions of DNAwere centrifuged, to prepare the precipitated DNA, which was thenseparated, dried and dissolved in 5 μl of water.

The thus prepared aqueous solution of said DNA fragment-β and theaqueous solution of the truncated plasmid DNA were mixed together ateach volume of 5 μl. The resulting mixture (10 μl) was subjected to aligation reaction by means of a DNA ligation kit (manufactured by TaKaRaBrewery Co., Ltd.), so that the two DNAs as contained in said resultingmixture were ligated with each other. The resulting ligation reactionmixture was added with a 1/10-fold volume of 3 M sodium acetate and a2-fold volume of ethanol, and the resulting admixture was incubated at−20° C. for about 6 hours. The resulting incubated reaction mixture wascentrifuged, to precipitate the DNA, which was then separated and dried.The thus recovered DNA was dissolved in 5 μl of water to prepare anaqueous solution of the DNA.

The DNA, which was present in the resulting aqueous solution soprepared, was a double-stranded recombinant plasmid (thepBluescript-DNA-158N plasmid as described above) which was prepared byligating the DNA fragment-β with said truncated plasmid as produced bythe cleavage of the plasmid vector pBluescript II SK(+) with KpnI andSacI, and in which the modified DNA-158N sequence was contained and waspresent in the inserted DNA region of said recombinant plasmid.

(ii) Further, Escherichia coli XL1-Blue MRF′ was transformed byintegrating said pBluescript-DNA-158N plasmid therein. Then, the sotransformed Escherichia coli cells were cultured in a liquid culturemedium.

From the cultured cells of the transformed Escherichia coli bacteria wasextracted a further amount of the plasmid. In this manner, therecombinant plasmid carrying therein the modified DNA-158N sequencecould be cloned.

(5) Recovery of DNA Fragment Carrying the Modified DNA-158N Sequence

10 μg of the plasmid DNA of the so obtained pBluescript-DNA-158N plasmidwas then digested with 10 units of XbaI and 10 units of SacI in a bufferM (manufactured by TaKaRa Brewery Co., Ltd.). The digestion reactionmixture obtained was fractionated by low-melting agaroseelectrophoresis. A DNA fragment (referred to as DNA fragment-β-2) whichcarries therein the DNA-158N sequence (of a length of 1143 bp), was cutout from the agarose gel. The agarose gel band containing the DNAfragment-β-2 was added with an equal volume of a TE buffer (containing10 mM Tris-HCl, pH 8 and 1 mM EDTA). The resulting mixture was heated at68° C. for 20 minutes, to dissolve the agarose in the TE buffer. Theresulting solution was extracted twice with aqueous saturated phenol, toremove the agarose. The resulting phenol extract containing the DNA wasadded with a 1/10-fold volume of 3 M sodium acetate and a 2-fold volumeof ethanol. The resulting mixture was incubated at −20° C. for about 6hours. The resulting incubated solution was centrifuged at 15,000 rpm at4° C. for 10 minutes. The resulting precipitated DNA was then driedunder reduced pressure. The thus recovered DNA powder was dissolved in10 μl of water. Said DNA powder comprised the DNA fragment-β-2 whichcarried therein the desired, modified DNA-158N sequence.

EXAMPLE 3

The present Example illustrates the preparation of a DNA fragmentcarrying therein the DNA sequence which is designated as the modifiedDNA-166A sequence (namely, the DNA sequence having the nucleotidesequence of SEQ ID No. 7 of Sequence Listing) and is produced inaccordance with the second aspect of the invention.

(1) Construction of Synthetic Oligonucleotide as Primer for PCR Method

A DNA synthesizer (Model-391; manufactured by Applied BioSystems, Co.)was used to produce by chemical synthesis a primer No. 4 which was theoligonucleotide having the nucleotide sequence shown in SEQ ID No. 10 ofSequence Listing.

(2) Preparation of Template for PCR Method

The vector pDAP8-1-XS, which is described above in the item (2) ofExample 2, carries therein the DNA fragment which has a sequence lengthof about 1200 bp and which contains therein the DNA sequence of SEQ IDNo. 1 of Sequence Listing (namely, the DHDPS-DNA-1143 sequence), andsaid DNA fragment has been inserted between the XbaI cleavage site andthe SacI cleavage site in the vector pDAP8-1-XS.

This recombinant vector pDAP8-1-XS was used as the template in thepresent Example 3 to carry out the following PCR procedures.

(3) Amplification of the Required DNA Fragment by PCR, and Recoverythereof

(i) First Step of PCR Method

In order to produce the required DNA fragment by amplification accordingto the PCR method, the following reactions (A) and (B) were conducted asthe first step of PCR method.

Thus, the reaction (A) comprised adding 5 μl of the recombinant plasmidvector pDAP8-1-XS for use as the template, as well as 1 μM of the primerFW-1, and 1 μM of the primer No. 4 of SEQ ID No. 10 to 100 μl of anamplification mixture [containing 10 mM Tris-HCl, pH 8.3, 1 mM MgCl₂, 50mM KCl, a mixture of 0.2 mM each of 4 dNTPs, and 2.5 units of LA Taq DNApolymerase], followed by effecting the amplification reaction. Theamplified DNA product so obtained is now referred to as DNA fragment-D.

Furthermore, the reaction (B) comprised adding 5 μl of the recombinantplasmid vector pDAP8-1-XS for use as the template, as well as 1 μM ofthe primer RV-1, and 1 μM of the BS KPN-1 to the amplification mixtureof the same formulation as in the reaction (A), followed by effectingthe amplification reaction. The amplified DNA product so produced by thereaction (B) is referred to as DNA fragment-B. The reaction (B) wasconducted in a similar way as in the item (3) of Example 2.

The above amplification reaction was promoted by repeating 30 times areaction cycle which consisted of three reaction procedures, namelydenaturation at 94° C. for 30 seconds, annealing at 55° C. for 2minutes, and extension at 72° C. for 2 minutes in a PCR reactor [ProgramTemp Control System PC-700; manufactured by Asthec, Co. Ltd.].

After the termination of the amplification reaction, the amplificationreaction mixture coming from the reaction (A) was fractionated bylow-melting agarose electrophoresis. And, a band containing the DNAfragment-D of 480 bp as the amplified DNA product was cut and separatedout of the agarose gel. Furthermore, the amplification reaction mixturecoming from the reaction (B) was fractionated by low-melting agaroseelectrophoresis. And, a band containing the DNA fragment-B of 1200 bp asthe amplified DNA product was cut and separated out of the agarose gel.

From these gel bands so cut out and separated, a purified product of theDNA fragment-D and a purified product of the DNA fragment-B wereseparated respectively by means of Geneclean II Kit (manufactured byFunakoshi Co. Ltd.) and then recovered.

(ii) Second Step of PCR Method

The second step of the PCR method comprised adding 1 μl of the primerRV-1 and 1 μl of the primer FW-1, as well as 1 μl of the DNA fragment-Dand 1 μl of the DNA fragment-B (which had been amplified individually bythe reactions (A) and (B), respectively) to 100 μl of an amplificationmixture [containing 10 mM Tris-HCl, pH 8.3, 1 mM MgCl₂, 50 mM KCl, amixture of 0.2 mM each of 4 dNTPs, and 2.5 units of LA Taq DNApolymerase], followed by effecting the amplification reaction. Thisamplification was then effected by repeating 20 times a reaction cyclewhich consisted of three reaction procedures, namely denaturation at 94°C. for 30 seconds, annealing at 55° C. for 2 minutes, and extension at72° C. for 2 minutes.

After the termination of the above amplification reaction the resultantamplification reaction solution was fractionated by low-melting agaroseelectrophoresis. Then, a band containing the desired DNA fragment-E of1200 bp as the amplified DNA product was cut out of the agarose gel.From the gel band so cut out, a purified product of the DNA fragment-Ewas separated and recovered by means of Geneclean II Kit (manufacturedby Funakoshi Co., Ltd.).

This DNA fragment-E is a DNA fragment of about 1350 bp, which carriestherein the DNA-166A sequence (a length of 1143 bp) having thenucleotide sequence of SEQ ID No. 7 of Sequence Listing, and which has aKpnI cleavage site at upstream of the 5′ terminus of the DNA-166Asequence and has an SacI cleavage site at downstream of the 3′ terminusof the DNA-166A sequence.

(4) Cloning of the DNA Fragment Carrying the Modified DNA-166A Sequence

The DNA fragment-E produced as above was used to produce the DNAfragment carrying the desired, modified DNA-166A sequence by cloning. Asufficient amount of said DNA fragment to be produced was obtainedaccording to the following procedures.

(i) 10 μl of the DNA fragment-E produced as above was first digestedwith 10 units each of restriction endonucleases KpnI and SacI in an Lbuffer (manufactured by TaKaRa Brewery Co., Ltd.). The DNA fragment asproduced by this digestion is now referred to as DNA fragments. Further,10 μl of the pBluescript II SK(+) was digested with 10 units each ofKpnI and SacI in an L buffer (manufactured by TaKaRa Brewery Co., Ltd.),to prepare a truncated plasmid. The resulting digested mixturecontaining the DNA fragment-γ, as well as the resulting digested mixturecontaining the truncated plasmid were added individually with a1/10-fold volume of 3 M sodium acetate and a 2-fold volume of ethanol.The resulting mixtures were each incubated at −20° C. for about 6 hours.Thereafter, the resulting individual incubated mixtures were eachcentrifuged, to produce the precipitated DNA, which was separated, driedand dissolved in 5 μl of water.

Thus, there were prepared the aqueous solution of the DNA fragment-γ, aswell as the aqueous solution of the truncated DNA. These two aqueoussolutions were mixed together at each volume of 5 μl. The resultingmixture (10 μl) was subjected to a ligation reaction with using a DNAligation kit (manufactured by TaKaRa Brewery Co., Ltd.), so that the twoDNAs contained in said resulting mixture were ligated with each other.To the resulting ligation reaction mixture were added a 1/10-fold volumeof 3 M sodium acetate and a 2-fold volume of ethanol. The resultingadmixture was incubated at −20° C. for about 6 hours. The resultingincubated admixture was centrifuged to precipitate the ligated DNA,which was then separated and dried. The thus recovered DNA was furtherdissolved in 5 μl of water to prepare an aqueous solution of said DNA.

The DNA which is contained in the resultant aqueous solution was adouble-stranded recombinant plasmid (that is, the pBluescript-DNA-166Aplasmid described hereinbefore) which was prepared by ligating said DNAfragment-γ with said truncated plasmid as produced by the cleavage ofthe plasmid vector pBluescript II SK(+) with KpnI and SacI, and in whichthe modified DNA-166A sequence was carried within the inserted DNAregion of the recombinant plasmid.

(ii) Escherichia coli XL1-Blue MRF′ was then transformed by integratingthe above-mentioned pBluescript-DNA-166A plasmid therein. Further, theso transformed Escherichia coli cells were cultured in a liquid culturemedium.

From the culture of the so transformed Escherichia coli cells was thenextracted the recombinant plasmid. In this manner, the recombinantplasmid carrying therein the modified DNA-166A sequence could be cloned.

(5) Recovery of the DNA Fragment Carrying therein the Modified DNA-166ASequence

10 μl of the plasmid DNA of the so produced pBluescript-DNA-166A plasmidwas then digested with 10 units of XbaI and 10 units of SacI in a bufferM (manufactured by TaKaRa Brewery Co., Ltd.). The resulting digestedmixture was fractionated by low-melting agarose electrophoresis. The gelband which contained therein the so produced DNA fragment-γ-2 carryingthe DNA-166A sequence (of a length of 1143 bp) was cut out of theagarose gel. The so cut-out agarose gel band containing the DNAfragment-γ-2 therein was added with an equal volume of a TE buffer(containing 10 mM Tris-HCl, pH 8 and 1 mM EDTA). The resulting mixturewas heated at 68° C. for 20 minutes, to dissolve the agarose. Theresulting solution of the agarose was extracted twice with aqueoussaturated phenol, to remove the agarose. The resulting phenol extractcontaining the DNA therein were added with a 1/10-fold volume of 3 Msodium acetate and a 2-fold volume of ethanol. The resulting mixture wasincubated at −20° C. for about 6 hours. The resulting incubated mixturewas centrifuged at 15,000 rpm at 4° C. for 10 minutes. The resultingprecipitated DNA was then separated and dried under reduced pressure.The so recovered powder of the DNA was dissolved in 10 μl of water. ThisDNA powder was formed of the DNA fragment-γ-2 of about 1200 bp carryingtherein the desired, modified DNA-166A sequence.

Further, in the same manner as described in Example 1, item (7)hereinbefore, the DNA fragment-β-2 which was produced in Example 2, item(5) herinbefore was examined by an automatic DNA sequencer, namely ALFDNA Sequencer II, with using of a nucleotide sequencing kit. It was thusconvinced that the said DNA fragment-β-2 was a DNA fragment whichcarried therein the modified DNA-158N sequence having the nucleotidesequence of SEQ ID No. 5 of Sequence Listing.

Equally, the said DNA fragment-γ-2 as produced in Example 3, item (5)above was examined by the nucleotide-sequencing experiments in the samemanner as described just above. The said DNA fragment-γ-2 was verifiedto be a DNA fragment which carried therein the modified DNA-166Asequence having the nucleotide sequence of SEQ ID No. 7 of SequenceListing.

EXAMPLE 4

The present Example illustrates a method for transforming a rice plant,which comprises introducing the DNA sequence of the first aspect of theinvention or the modified DNA sequence of the second aspect of theinvention, as an exogenous gene, into the rice plant.

(1) Construction of Recombinant Vector for use in the Introduction ofExogenous Gene

(i) The DNA (10 μl) of plasmid vector pBI221, which was of 5.7-kb-lengthand carried therein the 35S promoter of cauliflower mosaic virus, theNOS terminator and the ampicillin-resistant gene (manufactured byClontech, Co.), was digested with restriction endonucleases XbaI andSacI in a buffer M (manufactured by TaKaRa Brewery Co., Ltd.). From theresulting digestion mixture was precipitated the DNA, which wascollected by centrifugation and then dried. A vector fragment of about3.8 kb, which carried the 35S promoter and NOS terminator therein, wasthus harvested.

(ii) The vector fragment so obtained was dissolved in 5 μl of water. Theresulting aqueous solution (5 μl) of said vector fragment was mixed withan aqueous solution (5 μl) of the aforesaid DNA fragment-XS which was aDNA of about 1143 bp carrying therein the DHDPS-DNA-1143 sequence (of1143 bp) described in SEQ ID No. 1, and which was produced in Example 2hereinbefore according to the first aspect of the invention.

The resulting mixture was treated with a DNA ligation kit (manufacturedby TaKaRa Brewery Co., Ltd.), to effect ligation of the DNAS. In thismanner, there was prepared a cyclic recombinant vector which was formedby ligating said DNA fragment-XS with the XbaI/SacI-cleaved vectorfragment derived from the plasmid vector pBI221. The cyclic recombinantvector so prepared is hereinafter referred to as vector pDAP. Thisvector pDAP was of a 4.9-kb length and had such a structure where theDNA fragment-XS region thereof was inserted and ligated between the 35Spromoter region and the NOS terminator region of the vector pDAP and theampicillin-resistant gene (Am^(r)) was incorporated in the recombinantvector.

(iii) The DNA fragment-β-2 as produced in Example 2 (that is, the DNA ofa size of about 1143 bp, which carried therein the modified DNA-158Nsequence of 1143 bp described in SEQ ID No. 5 as provided in the secondaspect of the invention) was employed instead of the DNA fragment-XSproduced above in Example 2, to carry out the ligation reactionsimilarly to Example 4, (ii) above. Thereby, the DNA fragment-β-2 wasligated to the XbaI/SacI-cleaved vector fragment of the plasmid vectorpBI221 to prepare a cyclic recombinant vector, which is hereinafter isreferred to as vector p158N. This recombinant vector 158N was of a4.9-kb length and had such a structure that the DNA fragment-β-2 regionthereof was inserted and ligated between the 35S promoter region and theNOS terminator region of the recombinant vector.

(iv) Further, the DNA fragment-γ-2 as produced in Example 3 (that is,the DNA of a size of about 1143 bp, which carried therein the modifiedDNA-166A sequence of 1143 bp of described as SEQ ID No. 7 as provided inthe second aspect of the invention) was employed instead of the DNAfragment-XS produced in Example 2, to carry out the ligation reactionsimilarly. Thereby, the DNA fragment-γ-2 was ligated to theXbaI/SacI-cleaved vector fragment of the plasmid vector pBI221, toprepare a cyclic recombinant vector, which is now referred to as vectorp166A. This recombinant vector 166A was of a 4.9-kb length and had sucha structure that the DNA fragment-γ-2 region thereof was inserted andligated between the 35S promoter region and the NOS terminator region ofthe recombinant vector.

(2) Cloning of the Recombinant Vector

The aqueous solution (10 μl) of either the recombinant vector pDAP orthe recombinant vector p158N or the recombinant vector p166A obtained asabove (containing 10 mg of the DNA in the solution) and 100 μl of acommercially available Escherichia coli XL1-Blue MRF′ competent cellswere placed together in a 1.5-ml tube. The resulting mixture in the tubewas incubated on ice bath for 30 minutes, then at 42° C. for 30 seconds,and again on ice bath for 2 minutes. After this incubation, theincubated mixture was treated in the same manner as described in theitem (6) of Example 1 hereinbefore, followed by culturing thetransformed Escherichia coli cells under shaking.

The resulting culture of the transformed cells of Escherichia coli wasplated on an LB agar medium as supplemented with ampicillin and theother additives, in the same manner as described in the item (6) ofExample 1. The bacteria cells were cultured on the medium at 37° C. for16 hours.

In this way, there were obtained 10 colonies of the transformedEscherichia coli cell which was resistant to ampicillin and had beentransformed by the integration therein of the recombinant vector pDAP orthe recombinant vector 158N or the recombinant vector 166A. These tencolonies of the transformed Escherichia coli cell were proliferated in aliquid culture medium containing 50 mg/l of ampicillin.

(3) Recovery of the Recombinant Vector

From the so proliferated cells of the transformed Escherichia colibacteria present in each of the said 10 colonies, there were isolatedand purified the recombinant plasmids by means of a plasmid purificationkit (QIA Filter Plasmid Midi Kit; manufactured by QIAGEN, CO. LTD.).

The ten lots of the recombinant plasmids so obtained were digested withrestriction endonucleases XbaI and SacI, respectively. Then, theresulting digested DNA fragments were individually analyzed by agarosegel electrophoresis.

With making reference to the results of the above analysis, such arecombinant plasmid, which carried therein the DHDPS-DNA-1143 sequenceof the first aspect of the invention as normally inserted at downstreamof the 35S promoter region of the recombinant vector, was selected fromamong the recombinant plasmids as isolated in the above and then washarvested as the vector pDAP.

In the same manner as above, further such a recombinant plasmid whichcarried therein the modified DNA-158N sequence of the second aspect ofthe invention as normally inserted at downstream of the 35S promoterregion, was selected from among the recombinant plasmids as isolated inthe above, and then was harvested as the vector p158N.

In the same manner as above, still additionally, such a recombinantplasmid, which carried therein the modified DNA-166N sequence of thesecond aspect of the invention as normally inserted at downstream of the35S promoter region, was selected from among the isolated recombinantvectors and then was harvested as the vector p166A.

The Escherichia coli XL1-Blue MRF′ strain, which has been transformed bythe integration therein of the recombinant vector p158N as above, isdesignated as Escherichia coli XL1-Blue MRF′/p158N, and has beendeposited under Accession No. FERM BP-6323 in terms of the BudapestTreaty since Apr. 13, 1998 at the National Institute of Bioscience andHuman-Technology, the Agency of Industrial Science and Technology,supra., in Japan.

The Escherichia coli XL1-Blue MRF′ strain, which has been transformed bythe integration therein of the recombinant vector p166A as above, isdesignated as Escherichia coli XL1-Blue MRF′/p166A, and has beendeposited under Accession No. FERM BP-6324 in terms of the BudapestTreaty since Apr. 13, 1998 at the same National Institute, supra., inJapan.

(4) Preparation of Rice Callus

The ripe seeds of completely fructifying rice (variety: Nipponbare) werehulled. The resulting hulled rice seeds with outer skin were sterilizedby sequential immersions thereof in an aqueous 70% ethanol solution for60 seconds and then in a dilute aqueous solution containing sodiumhypochlorite at an effective chlorine content of about 1%, for 6minutes. The sterilized rice seeds were rinsed in sterile water.

In an MS medium containing the usual inorganic components and furthersupplemented with 30 g/liter of sucrose, a plant hormone of 2,4-PA of 2mg/liter and 8 g/liter of agar, there were placed the above, sterilizedrice seeds. The rice seeds were then incubated at 28° C. underirradiation of light at 2000 lux for 16 hours per day for 45 days. Thecallus was thus formed from the seed. The calluses were cut off out ofthe albumen part of the incubated seeds, followed by sieving with astainless-mesh sieve of a pore size of 1 mm. There were obtainedcalluses each of a dimension of 1 mm or less at a cell amount of 3 ml interm of PCV (packed cell volume: the quantity of compressed cells).

(5) Preparation of Whiskers

Commercially available whiskers (LS20; manufactured by TitaniumIndustry, Co., Ltd.) made of potassium titanate were placed in a tubularcontainer of 1.5 ml capacity and then sterilized overnight in 0.5 ml ofethanol as add in the small tubular container. The ethanol was removedcompletely by evaporation, and the sterilized whiskers were recovered.Sterile water was placed in the tubular container containing thesterilized whiskers therein, and the contents in the tube; wereagitated. The whiskers and the sterile water together were centrifuged,and the supernatant water was discarded. The whiskers were thus rinsed.The whiskers-rinsing procedure was repeated three times. Thereafter, 0.5ml of the R2 liquid medium was added to the tubular container, toprepare a suspension of the whiskers in the medium.

(6) Preparation of Materials to be Used for Introduction of ExogenousGene into Rice Callus Cell

The recombinant vector pDAP, or the recombinant vector p158N, or therecombinant vector p166A, which were prepared as described above in theitem (3) of Example 4, was dissolved at a concentration of 1 mg/ml in aTE buffer (comprising 10 mM Tris-HCl, 1 mM EDTA, pH 8), to prepare asolution of the recombinant vector.

In the tubular container containing therein the suspension of thewhiskers, there were charged 250 μl of the calluses each of a dimensionof 1 mm or less. The resulting mixture in the container was thenagitated together and subsequently centrifuged at 1,000 rpm for 10seconds, to precipitate the calluses and whiskers. The resultingsupernatant was discarded, and a mixture of the rice callus cells andthe whiskers was afforded.

To the mixture of the callus cells and the whiskers present in the tube,there were added 10 μl (with a DNA content of 10 μl) of the saidrecombinant vector (namely, the vector PDAP or the vector p158N or thevector p166A) and 10 μl (with a DNA content of 10 μl) of a plasmidvector p35SC-SS which carried therein a gene resistant to a herbicide,phosphinothricin, as the selection marker (see JP-A-8-154513). Aftersufficient shaking, the resultant homogenous mixture was recovered.

The tube containing therein said homogenous mixture was centrifuged at18,000×g for 5 minutes. After this centrifugation, the mixture containedin the tube was again shaken together. This centrifugation procedure andre-shaking procedure were individually repeated three times.

(7) Manipulation for Introduction of the Exogenous Gene into Rice Callus

The tube, which was containing therein the resultant homogenous mixturecomprising the callus cells, the whiskers, and the recombinant vectorcarrying therein the DNA of this invention and also the plasmid vectorp35SC-SS, prepared as above, was placed in the bath vessel of anultrasonic generator, so that the whole tube was sinked within the bath.The ultrasonic wave at a frequency of 40 kHz was irradiated onto thetube at an intensity of 0.25 W/cm² for one minute. After thisirradiation, the so ultrasonically treated mixture was incubated at 4°C. for 30 minutes.

The mixture thus treated ultrasonically was then rinsed with the R2liquid medium, and there were afforded the desired, transformed calluscells which carried said recombinant vector as introduced therein.

(8) Selection of the Callus Cells as Transformed by the IntroducedVector

The resultant calluses having the transformed cells in which therecombinant vector was introduced as above, were placed in a 3.5-cmpetri dish, to which was then added a volume of the R2 liquid mediumcontaining the usual inorganic components and further supplemented with30 g/liter of sucrose and 2 mg/liter of 2,4-PA. Subsequently, thetransformed callus cells were cultivated at 28° C. under shaking on arotary shaker (at 50 rpm.) and under irradiation of light at 2,000 luxfor 16 hours per day, to produce the divided callus cells through thecell-division.

On the 3^(rd) day of cultivation, the resultant suspension (3 ml) of thedivided cells was spread evenly on a medium which had been prepared byadmixing the N6 medium containing the usual inorganic components, with30 g/liter of sucrose, 2 mg/liter of 2,4-PA, 3 g/liter of Gelrite and 30mg/liter of phosphinothricin. Then, the cells on the medium werecultured at 28° C. under irradiation of light at 2000 lux for 16 hoursper day, for consecutive 30 days. The phosphinothricin-resistanttransformed callus cells were produced and obtained thereby.

(9) Re-selection of the Transgenic Callus Cell as Taransformed by theVector p158N or p166A

From among the phosphinothricin-resistant transgenic cultured calluscells so obtained, there was re-selected only such a desired culturedcells which had been transformed by the introduction of a sufficientamount of the DNA of the second aspect of this invention as theexogenous gene. For this purpose, 400 calluses composed of saidtransformed callus cells (each having a 2-mm diameter) were transplantedonto a culture medium which was prepared by admixing the N6 medium ofthe inorganic component composition with 30 g/liter of sucrose, 2mg/liter of 2,4-PA, 3 g/liter of Gelrite and 200 mg/liter of alysine-analog, S-(2-aminoethyl)cysteine (referred to as “AEC”hereinafter) usable as a cell-proliferation-inhibitor. The callusescomposed of the transformed callus cells so transplanted were culturedat 28° C. under irradiation of light at 2,000 lux for 16 hours per day,for consecutive 30 days. The calluses composed of the transformed calluscells carrying therein the vector p158N or p166A, which could grow inthe culture medium containing the added AEC, were re-selected in theabove manner.

(10) Regeneration of Plant from the Transformed Callus Cells soRe-selected

98 to 100 calluses (each of a 5-mm diameter) comprising thephosphinothricin-resistant and AEC-resistant transformed callus cells asobtained in the above were transplanted onto a culture medium which wasprepared by admixing the MS medium of the inorganic componentcomposition with 30 g/liter of sucrose, 2 mg/liter of benzyladenine, 1mg/liter of naphthalene acetate, and 3 g/liter of Gelrite. The callusescomposed of the trans-formed callus cells so transplanted were thencultured at 28° C. under irradiation of light at 2,000 lux for 16 hoursper day, for consecutive 30 days. From the calluses composed of thetransformed callus cells so cultured were regenerated the bud and rootof the rice plant. The plumules having the regenerated buds and rootswere grown to a height of 10 to 30 mm, and the grown plumules weretransplanted into an MS medium which was further supplemented with 30g/liter of sucrose and 3 g/liter of Gelrite and which was placed in atest tube of a diameter of 45 mm and a length of 25 cm. The transplantedplumules were cultivated therein for 20 days, to afford the transgenicrice plants.

By conducting the aforementioned method, 80 plants of the transgenicrice plant could be regenerated from 98 calluses composed of thetransformed callus cells which carried therein the DHDPS-DNA-1143sequence of the first aspect of the invention as the exogenous gene.Further, 76 plants of the transgenic rice plant could be regeneratedfrom 100 calluses composed of the AEC-resistant transformed callus cellswhich carried therein the modified DNA-158N sequence of the secondaspect of the invention as the exogenous gene.

Furthermore, 79 plants of the transgenic rice plant could be regeneratedfrom 100 calluses composed of the AEC-resistant transformed callus cellswhich carried therein the modified DNA-166N sequence of the secondaspect of the invention as the exogenous gene.

(11) Genetic Analysis of the Regenerated Plant Body of the TransgenicRice Plant

The DNA for encoding the DHDPS present in the so regenerated transgenicrice plant was analyzed by PCR method according to the followingprocedure.

(i) From the regenerated transgenic rice plant as produced in the aboveitem (10) were collected the leaves. 50 mg of such leaves was placed ina 1.5-ml capacity microtube, to which was then added 300 μl of 20 mMTris-HCl buffer (pH 7.5) containing 10 mM EDTA. The leaves in the bufferwere disrupted. To the disrupted leaves was added 20 μl of 20% SDS,followed by heating at 65° C. for 10 minutes. The resulting mixture wasadded with 100 μl of 5M potassium acetate and the resulting admixturewas left to stand on ice bath for 20 minutes and then centrifuged at acentrifugal acceleration rate of 17,000×g for 20 minutes. 200 μl ofisopropanol was added to the resulting supernatant. The resultingmixture in the tube was agitated by tumbling of the tube. The agitatedmixture was then centrifuged at centifugal acceleration rate of 17,000×gfor 20 minutes. The resulting precipitated DNA was separated and driedunder reduced pressure. The DNA so obtained was dissolved in 100 μl of aTE buffer.

(ii) Further, an oligonucleotide of the nucleotide sequence of SEQ IDNo. 14 of Sequence Listing and an oligonucleotide of the nucleotidesequence of SEQ ID No. 15 of Sequence Listing were prepared as primersfor PCR.

5 μl of the above-mentioned DNA derived from the regenerated transgenicrice plant was used as template, and 1 μM each of the above-mentionedtwo types of the oligonucleotides were used as primers. They were thenadded to 100 μl of an amplification mixture [containing 10 mM Tris-HCl,pH 8.3, 1.0 mM MgCl₂, 50 mM KCl, 0.01% gelatin, pH 8.3, a mixture of 4dNTPs each at 0.2 mM, and a Taq DNA polymerase of 2.5 units], followedby effecting the amplification reaction of DNA. The amplificationmixture herein used had been prepared by using a PCR kit (PCRAmplification Kit; manufactured by TaKaRa Brewery, Co., Ltd.).

The amplification reaction was effected by repeating 30 times a reactioncycle which consisted of three reaction procedures, namely denaturationat 94° C. for one minute, annealing at 60° C. for 30 seconds andextension at 72° C. for one minute.

(iii) The resulting PCR reaction solution was then subjected to agaroseelectrophoresis in a conventional manner. Thereby, detection could bemade of various fragments of DNAs which had been amplified from the DNAsas extracted from the regenerated transgenic rice plant.

Further analysis of the sequences of the resultant various DNA fragmentswas conducted by means of a nucleotide sequencing kit, to verify that aDNA fragment, which was corresponding to the DHDPS-DNA-1143 sequence orthe modified DNA-158N or the modified DNA-166A sequence of thisinvention, was present in the said resultant various DNA fragments whichwere extracted from the regenerated transgenic rice plant in the abovemanner.

By conducting the genetic analysis as above by PCR, it could beconfirmed that there were produced the regenerated transgenic riceplants which necessarily carried therein the introduced exogenous gene.The regenerated rice plants so confirmed were then transplanted andcultivated in pot containing therein cultivation soil. These regeneratedrice plants so transplanted could normally grow further so that theirself-fertilized seeds could be yielded and recovered.

(12) Assay of Lysine Content of the Regenerated Plant Body of TransgenicRice Plant

Green leaves were harvested respectively from the regenerated transgenicrice plant having the plant cells which carried the introducedrecombinant vector pDAP containing therein the DHDPS-DNA-1143 sequenceof the first aspect of the invention, and also from the regeneratedtransformant rice plant having the plant cells which carried theintroduced recombinant vector pDNA-158N containing therein the DNA-158Nsequence of the second aspect of the invention, as well as from theregenerated transgenic rice plant having the plant cells which carriedthe introduced recombinant vector pDNA-166A containing therein theDNA-166A sequence of the second aspect of the invention.

One gram each of the green leaves harvested individually from thetransgenic rice plants was placed in a first tube of a 1.5-ml capacity,to which was added then 1 ml of 50% acetonitrile, followed by disruptingthese leaves. The disputed leave mixture so obtained was transferred toa second tube of a 1.5 ml capacity, and was centrifuged at a centrifugalacceleration rate of 17,000×g for 20 minutes. The resulting supernatantwas transferred in a third tube, to which was then added 1 ml of 50%acetonitrile. The mixture in the third tube was agitated by invertingthe tube upside down, followed by re-centrifugation of the mixture. Theresulting supernatant was added to the first tube. These procedures wererepeated three times. In this manner, there was prepared a solution inacetonitrile of lysine which was extracted and recovered from the greenleaves.

The acetonitrile solution of lysine so extracted was absolutelyevaporated to dryness under reduced pressure. The solid residue wasadded with 1 ml of distilled water, to prepare an aqueous solutionthereof. Each of the aqueous solutions so prepared was centrifuged at17,000×g for 20 minutes, to afford the supernatant of 0.5 ml. With 100μl of each supernatant so obtained was mixed 100 μl of 5 mM DNFB(2,4-dinitro-1-fluorobenzene). The resulting mixture was incubatedovernight. To each of the resulting incubation mixtures was added 200 μlof acetonitrile, followed by agitation and centrifugation at 17,000×gfor 20 minutes. Thereby, an extract solution of lysine was recovered asthese resulting supernatant solutions. The resulting extract solutionswere individually subjected to high-performance liquid chromatography(HPLC) device (Type 8020; manufactured by Tosoh, Co., Ltd.), todetermine the free lysine content in each extract. The column used inthis HPLC was CAPCELL PAK-C18 (manufactured by Shiseido, Co., Ltd.). TheHPLC was conducted with using acetonitrile/water as the developmentsolvent with a concentration gradient of acetonitrile of from 60% to 72%and at the flow of 0.8 ml/min. The lysine content was determined bymeasurement of the absorbance of light at 350 nm.

As a control rice plant was used a plant of an ordinary rice plant(variety: Nipponbare). The results of the determination as obtained areshown in Table 1 below.

TABLE 1 Content of free lysine rice plant tested (n mol/FW g) Controlrice plant 59 rice plant carrying vector 360 pDAP rice plant-1 carrying688 vector p158N rice plant-2 carrying 652 vector p158N rice plant-1carrying 673 vector p166A

As will be clear from the results shown in Table 1, it is confirmed thatthe content of the free lysine produciable in the rice plant can beelevated by introducing the novel DNA sequences as an exogenous geneinto the rice plant, with using a recombinant vectors which carries apromoter capable of expressing in the plant cell.

Industrial Utilizability

As will be obvious from the foregoing descriptions, this inventionprovides such novel DNA sequences for encoding the dihydrodipicolinatesynthase of rice. By introducing the DNA sequences of this invention asthe exogenous gene in a rice plant, it is made feasible to increase thecontent of lysine which is one of the essential amino acids in the riceplant. The DNA sequences provided in accordance with this invention canbe introduced as an exogenous gene into the rice plant and into otheruseful plants such as corn, soybean, wheat and barley by a conventionaland known biotechnological method. Thus, the DNA sequences provided inaccordance with this invention are useful for cultivation of novelvarieties of plant which are capable of generating seeds of high lysinecontent.

15 1 1143 DNA Oryza sativa CDS (1)..(1143) 1 atg gcg tcg ctg ctg atc gccagc acg ggg ggc tgc cca ccg cct cgc 48 Met Ala Ser Leu Leu Ile Ala SerThr Gly Gly Cys Pro Pro Pro Arg 1 5 10 15 gtg gaa gga cgc cgc cgc cctggg acc cgc tcc ggc ttg gcg cga cct 96 Val Glu Gly Arg Arg Arg Pro GlyThr Arg Ser Gly Leu Ala Arg Pro 20 25 30 tgg ccc gcc gcc gtg gct gca ccggcg ccg ctg ctc agg att agc aga 144 Trp Pro Ala Ala Val Ala Ala Pro AlaPro Leu Leu Arg Ile Ser Arg 35 40 45 gga aag ttt gca ttg cag gcc atc accctt gat gat tat ctt cca atg 192 Gly Lys Phe Ala Leu Gln Ala Ile Thr LeuAsp Asp Tyr Leu Pro Met 50 55 60 cga agt act gaa gtg aaa aat cgg aca tcaaca gct gat atc act agt 240 Arg Ser Thr Glu Val Lys Asn Arg Thr Ser ThrAla Asp Ile Thr Ser 65 70 75 80 ctc aga gta att aca gcg gtc aaa acc ccatat ctg cct gat gga aga 288 Leu Arg Val Ile Thr Ala Val Lys Thr Pro TyrLeu Pro Asp Gly Arg 85 90 95 ttt gat ctc gaa gca tat gat tca ctg ata aatatg cag ata gat ggt 336 Phe Asp Leu Glu Ala Tyr Asp Ser Leu Ile Asn MetGln Ile Asp Gly 100 105 110 ggt gct gaa ggt gta ata gtt gga gga aca acagga gag ggc cac ctt 384 Gly Ala Glu Gly Val Ile Val Gly Gly Thr Thr GlyGlu Gly His Leu 115 120 125 atg agc tgg gat gaa cac atc atg ctt att ggacat act gtt aac tgc 432 Met Ser Trp Asp Glu His Ile Met Leu Ile Gly HisThr Val Asn Cys 130 135 140 ttt ggt gct aaa gtt aaa gtg gta ggc aac acaggt agt aac tca aca 480 Phe Gly Ala Lys Val Lys Val Val Gly Asn Thr GlySer Asn Ser Thr 145 150 155 160 aga gag gct att cat gca aca gag cag ggattt gct gta ggt atg cat 528 Arg Glu Ala Ile His Ala Thr Glu Gln Gly PheAla Val Gly Met His 165 170 175 gcg gct ctc cat atc aat cct tac tat gggaag acc tct atc gaa ggg 576 Ala Ala Leu His Ile Asn Pro Tyr Tyr Gly LysThr Ser Ile Glu Gly 180 185 190 ttg ata tct cat ttt gag gct gtc ctc ccaatg ggt cca acc att att 624 Leu Ile Ser His Phe Glu Ala Val Leu Pro MetGly Pro Thr Ile Ile 195 200 205 tac aat gtt cca tct agg act ggc cag gatatt cct cct gca gtt att 672 Tyr Asn Val Pro Ser Arg Thr Gly Gln Asp IlePro Pro Ala Val Ile 210 215 220 gag gct gtt tca agt ttc aca aac ttg gcaggt gtg aaa gaa tgt gtt 720 Glu Ala Val Ser Ser Phe Thr Asn Leu Ala GlyVal Lys Glu Cys Val 225 230 235 240 gga cat gag agg gtt aag tgc tac actgac aaa ggt ata acc ata tgg 768 Gly His Glu Arg Val Lys Cys Tyr Thr AspLys Gly Ile Thr Ile Trp 245 250 255 agt ggt aat gat gat gaa tgc cat gattct agg tgg aaa tat ggt gcc 816 Ser Gly Asn Asp Asp Glu Cys His Asp SerArg Trp Lys Tyr Gly Ala 260 265 270 act gga gtt att tct gtg gct agc aacctt att cct ggt ctc atg cac 864 Thr Gly Val Ile Ser Val Ala Ser Asn LeuIle Pro Gly Leu Met His 275 280 285 gat ctc atg tat gaa ggg gag aat aagacg cta aat gag aag ctc ttt 912 Asp Leu Met Tyr Glu Gly Glu Asn Lys ThrLeu Asn Glu Lys Leu Phe 290 295 300 ccc ctg atg aaa tgg ttg ttt tgc cagcca aat cca att gct ctc aac 960 Pro Leu Met Lys Trp Leu Phe Cys Gln ProAsn Pro Ile Ala Leu Asn 305 310 315 320 act gcc ctg gct cag ctt gga gtggta agg cct gtt ttc aga tta cca 1008 Thr Ala Leu Ala Gln Leu Gly Val ValArg Pro Val Phe Arg Leu Pro 325 330 335 tat gta cct ctt cct ctt gaa aagagg gta gag ttt gtc cga atc gtt 1056 Tyr Val Pro Leu Pro Leu Glu Lys ArgVal Glu Phe Val Arg Ile Val 340 345 350 gaa tct att gga cgg gaa aac tttgtg ggt gag aac gag gca cgg gtt 1104 Glu Ser Ile Gly Arg Glu Asn Phe ValGly Glu Asn Glu Ala Arg Val 355 360 365 ctt gac gac gat gat ttt gtg ttggtc agt agg tac taa 1143 Leu Asp Asp Asp Asp Phe Val Leu Val Ser Arg Tyr370 375 380 2 380 PRT Oryza sativa 2 Met Ala Ser Leu Leu Ile Ala Ser ThrGly Gly Cys Pro Pro Pro Arg 1 5 10 15 Val Glu Gly Arg Arg Arg Pro GlyThr Arg Ser Gly Leu Ala Arg Pro 20 25 30 Trp Pro Ala Ala Val Ala Ala ProAla Pro Leu Leu Arg Ile Ser Arg 35 40 45 Gly Lys Phe Ala Leu Gln Ala IleThr Leu Asp Asp Tyr Leu Pro Met 50 55 60 Arg Ser Thr Glu Val Lys Asn ArgThr Ser Thr Ala Asp Ile Thr Ser 65 70 75 80 Leu Arg Val Ile Thr Ala ValLys Thr Pro Tyr Leu Pro Asp Gly Arg 85 90 95 Phe Asp Leu Glu Ala Tyr AspSer Leu Ile Asn Met Gln Ile Asp Gly 100 105 110 Gly Ala Glu Gly Val IleVal Gly Gly Thr Thr Gly Glu Gly His Leu 115 120 125 Met Ser Trp Asp GluHis Ile Met Leu Ile Gly His Thr Val Asn Cys 130 135 140 Phe Gly Ala LysVal Lys Val Val Gly Asn Thr Gly Ser Asn Ser Thr 145 150 155 160 Arg GluAla Ile His Ala Thr Glu Gln Gly Phe Ala Val Gly Met His 165 170 175 AlaAla Leu His Ile Asn Pro Tyr Tyr Gly Lys Thr Ser Ile Glu Gly 180 185 190Leu Ile Ser His Phe Glu Ala Val Leu Pro Met Gly Pro Thr Ile Ile 195 200205 Tyr Asn Val Pro Ser Arg Thr Gly Gln Asp Ile Pro Pro Ala Val Ile 210215 220 Glu Ala Val Ser Ser Phe Thr Asn Leu Ala Gly Val Lys Glu Cys Val225 230 235 240 Gly His Glu Arg Val Lys Cys Tyr Thr Asp Lys Gly Ile ThrIle Trp 245 250 255 Ser Gly Asn Asp Asp Glu Cys His Asp Ser Arg Trp LysTyr Gly Ala 260 265 270 Thr Gly Val Ile Ser Val Ala Ser Asn Leu Ile ProGly Leu Met His 275 280 285 Asp Leu Met Tyr Glu Gly Glu Asn Lys Thr LeuAsn Glu Lys Leu Phe 290 295 300 Pro Leu Met Lys Trp Leu Phe Cys Gln ProAsn Pro Ile Ala Leu Asn 305 310 315 320 Thr Ala Leu Ala Gln Leu Gly ValVal Arg Pro Val Phe Arg Leu Pro 325 330 335 Tyr Val Pro Leu Pro Leu GluLys Arg Val Glu Phe Val Arg Ile Val 340 345 350 Glu Ser Ile Gly Arg GluAsn Phe Val Gly Glu Asn Glu Ala Arg Val 355 360 365 Leu Asp Asp Asp AspPhe Val Leu Val Ser Arg Tyr 370 375 380 3 25 DNA Oryza sativa 3gtaatagttg gaggaacaac aggag 25 4 24 DNA Oryza sativa 4 gagctgagccagagcagtgt tgag 24 5 1143 DNA Oryza sativa CDS (1)..(1143) 5 atg gcg tcgctg ctg atc gcc agc acg ggg ggc tgc cca ccg cct cgc 48 Met Ala Ser LeuLeu Ile Ala Ser Thr Gly Gly Cys Pro Pro Pro Arg 1 5 10 15 gtg gaa ggacgc cgc cgc cct ggg acc cgc tcc ggc ttg gcg cga cct 96 Val Glu Gly ArgArg Arg Pro Gly Thr Arg Ser Gly Leu Ala Arg Pro 20 25 30 tgg ccc gcc gccgtg gct gca ccg gcg ccg ctg ctc agg att agc aga 144 Trp Pro Ala Ala ValAla Ala Pro Ala Pro Leu Leu Arg Ile Ser Arg 35 40 45 gga aag ttt gca ttgcag gcc atc acc ctt gat gat tat ctt cca atg 192 Gly Lys Phe Ala Leu GlnAla Ile Thr Leu Asp Asp Tyr Leu Pro Met 50 55 60 cga agt act gaa gtg aaaaat cgg aca tca aca gct gat atc act agt 240 Arg Ser Thr Glu Val Lys AsnArg Thr Ser Thr Ala Asp Ile Thr Ser 65 70 75 80 ctc aga gta att aca gcggtc aaa acc cca tat ctg cct gat gga aga 288 Leu Arg Val Ile Thr Ala ValLys Thr Pro Tyr Leu Pro Asp Gly Arg 85 90 95 ttt gat ctc gaa gca tat gattca ctg ata aat atg cag ata gat ggt 336 Phe Asp Leu Glu Ala Tyr Asp SerLeu Ile Asn Met Gln Ile Asp Gly 100 105 110 ggt gct gaa ggt gta ata gttgga gga aca aca gga gag ggc cac ctt 384 Gly Ala Glu Gly Val Ile Val GlyGly Thr Thr Gly Glu Gly His Leu 115 120 125 atg agc tgg gat gaa cac atcatg ctt att gga cat act gtt aac tgc 432 Met Ser Trp Asp Glu His Ile MetLeu Ile Gly His Thr Val Asn Cys 130 135 140 ttt ggt gct aaa gtt aaa gtggta ggc aac aca ggt agt atc tca aca 480 Phe Gly Ala Lys Val Lys Val ValGly Asn Thr Gly Ser Ile Ser Thr 145 150 155 160 aga gag gct att cat gcaaca gag cag gga ttt gct gta ggt atg cat 528 Arg Glu Ala Ile His Ala ThrGlu Gln Gly Phe Ala Val Gly Met His 165 170 175 gcg gct ctc cat atc aatcct tac tat ggg aag acc tct atc gaa ggg 576 Ala Ala Leu His Ile Asn ProTyr Tyr Gly Lys Thr Ser Ile Glu Gly 180 185 190 ttg ata tct cat ttt gaggct gtc ctc cca atg ggt cca acc att att 624 Leu Ile Ser His Phe Glu AlaVal Leu Pro Met Gly Pro Thr Ile Ile 195 200 205 tac aat gtt cca tct aggact ggc cag gat att cct cct gca gtt att 672 Tyr Asn Val Pro Ser Arg ThrGly Gln Asp Ile Pro Pro Ala Val Ile 210 215 220 gag gct gtt tca agt ttcaca aac ttg gca ggt gtg aaa gaa tgt gtt 720 Glu Ala Val Ser Ser Phe ThrAsn Leu Ala Gly Val Lys Glu Cys Val 225 230 235 240 gga cat gag agg gttaag tgc tac act gac aaa ggt ata acc ata tgg 768 Gly His Glu Arg Val LysCys Tyr Thr Asp Lys Gly Ile Thr Ile Trp 245 250 255 agt ggt aat gat gatgaa tgc cat gat tct agg tgg aaa tat ggt gcc 816 Ser Gly Asn Asp Asp GluCys His Asp Ser Arg Trp Lys Tyr Gly Ala 260 265 270 act gga gtt att tctgtg gct agc aac ctt att cct ggt ctc atg cac 864 Thr Gly Val Ile Ser ValAla Ser Asn Leu Ile Pro Gly Leu Met His 275 280 285 gat ctc atg tat gaaggg gag aat aag acg cta aat gag aag ctc ttt 912 Asp Leu Met Tyr Glu GlyGlu Asn Lys Thr Leu Asn Glu Lys Leu Phe 290 295 300 ccc ctg atg aaa tggttg ttt tgc cag cca aat cca att gct ctc aac 960 Pro Leu Met Lys Trp LeuPhe Cys Gln Pro Asn Pro Ile Ala Leu Asn 305 310 315 320 act gcc ctg gctcag ctt gga gtg gta agg cct gtt ttc aga tta cca 1008 Thr Ala Leu Ala GlnLeu Gly Val Val Arg Pro Val Phe Arg Leu Pro 325 330 335 tat gta cct cttcct ctt gaa aag agg gta gag ttt gtc cga atc gtt 1056 Tyr Val Pro Leu ProLeu Glu Lys Arg Val Glu Phe Val Arg Ile Val 340 345 350 gaa tct att ggacgg gaa aac ttt gtg ggt gag aac gag gca cgg gtt 1104 Glu Ser Ile Gly ArgGlu Asn Phe Val Gly Glu Asn Glu Ala Arg Val 355 360 365 ctt gac gac gatgat ttt gtg ttg gtc agt agg tac taa 1143 Leu Asp Asp Asp Asp Phe Val LeuVal Ser Arg Tyr 370 375 380 6 380 PRT Oryza sativa 6 Met Ala Ser Leu LeuIle Ala Ser Thr Gly Gly Cys Pro Pro Pro Arg 1 5 10 15 Val Glu Gly ArgArg Arg Pro Gly Thr Arg Ser Gly Leu Ala Arg Pro 20 25 30 Trp Pro Ala AlaVal Ala Ala Pro Ala Pro Leu Leu Arg Ile Ser Arg 35 40 45 Gly Lys Phe AlaLeu Gln Ala Ile Thr Leu Asp Asp Tyr Leu Pro Met 50 55 60 Arg Ser Thr GluVal Lys Asn Arg Thr Ser Thr Ala Asp Ile Thr Ser 65 70 75 80 Leu Arg ValIle Thr Ala Val Lys Thr Pro Tyr Leu Pro Asp Gly Arg 85 90 95 Phe Asp LeuGlu Ala Tyr Asp Ser Leu Ile Asn Met Gln Ile Asp Gly 100 105 110 Gly AlaGlu Gly Val Ile Val Gly Gly Thr Thr Gly Glu Gly His Leu 115 120 125 MetSer Trp Asp Glu His Ile Met Leu Ile Gly His Thr Val Asn Cys 130 135 140Phe Gly Ala Lys Val Lys Val Val Gly Asn Thr Gly Ser Ile Ser Thr 145 150155 160 Arg Glu Ala Ile His Ala Thr Glu Gln Gly Phe Ala Val Gly Met His165 170 175 Ala Ala Leu His Ile Asn Pro Tyr Tyr Gly Lys Thr Ser Ile GluGly 180 185 190 Leu Ile Ser His Phe Glu Ala Val Leu Pro Met Gly Pro ThrIle Ile 195 200 205 Tyr Asn Val Pro Ser Arg Thr Gly Gln Asp Ile Pro ProAla Val Ile 210 215 220 Glu Ala Val Ser Ser Phe Thr Asn Leu Ala Gly ValLys Glu Cys Val 225 230 235 240 Gly His Glu Arg Val Lys Cys Tyr Thr AspLys Gly Ile Thr Ile Trp 245 250 255 Ser Gly Asn Asp Asp Glu Cys His AspSer Arg Trp Lys Tyr Gly Ala 260 265 270 Thr Gly Val Ile Ser Val Ala SerAsn Leu Ile Pro Gly Leu Met His 275 280 285 Asp Leu Met Tyr Glu Gly GluAsn Lys Thr Leu Asn Glu Lys Leu Phe 290 295 300 Pro Leu Met Lys Trp LeuPhe Cys Gln Pro Asn Pro Ile Ala Leu Asn 305 310 315 320 Thr Ala Leu AlaGln Leu Gly Val Val Arg Pro Val Phe Arg Leu Pro 325 330 335 Tyr Val ProLeu Pro Leu Glu Lys Arg Val Glu Phe Val Arg Ile Val 340 345 350 Glu SerIle Gly Arg Glu Asn Phe Val Gly Glu Asn Glu Ala Arg Val 355 360 365 LeuAsp Asp Asp Asp Phe Val Leu Val Ser Arg Tyr 370 375 380 7 1143 DNA Oryzasativa CDS (1)..(1143) 7 atg gcg tcg ctg ctg atc gcc agc acg ggg ggc tgccca ccg cct cgc 48 Met Ala Ser Leu Leu Ile Ala Ser Thr Gly Gly Cys ProPro Pro Arg 1 5 10 15 gtg gaa gga cgc cgc cgc cct ggg acc cgc tcc ggcttg gcg cga cct 96 Val Glu Gly Arg Arg Arg Pro Gly Thr Arg Ser Gly LeuAla Arg Pro 20 25 30 tgg ccc gcc gcc gtg gct gca ccg gcg ccg ctg ctc aggatt agc aga 144 Trp Pro Ala Ala Val Ala Ala Pro Ala Pro Leu Leu Arg IleSer Arg 35 40 45 gga aag ttt gca ttg cag gcc atc acc ctt gat gat tat cttcca atg 192 Gly Lys Phe Ala Leu Gln Ala Ile Thr Leu Asp Asp Tyr Leu ProMet 50 55 60 cga agt act gaa gtg aaa aat cgg aca tca aca gct gat atc actagt 240 Arg Ser Thr Glu Val Lys Asn Arg Thr Ser Thr Ala Asp Ile Thr Ser65 70 75 80 ctc aga gta att aca gcg gtc aaa acc cca tat ctg cct gat ggaaga 288 Leu Arg Val Ile Thr Ala Val Lys Thr Pro Tyr Leu Pro Asp Gly Arg85 90 95 ttt gat ctc gaa gca tat gat tca ctg ata aat atg cag ata gat ggt336 Phe Asp Leu Glu Ala Tyr Asp Ser Leu Ile Asn Met Gln Ile Asp Gly 100105 110 ggt gct gaa ggt gta ata gtt gga gga aca aca gga gag ggc cac ctt384 Gly Ala Glu Gly Val Ile Val Gly Gly Thr Thr Gly Glu Gly His Leu 115120 125 atg agc tgg gat gaa cac atc atg ctt att gga cat act gtt aac tgc432 Met Ser Trp Asp Glu His Ile Met Leu Ile Gly His Thr Val Asn Cys 130135 140 ttt ggt gct aaa gtt aaa gtg gta ggc aac aca ggt agt aac tca aca480 Phe Gly Ala Lys Val Lys Val Val Gly Asn Thr Gly Ser Asn Ser Thr 145150 155 160 aga gag gct att cat gta aca gag cag gga ttt gct gta ggt atgcat 528 Arg Glu Ala Ile His Val Thr Glu Gln Gly Phe Ala Val Gly Met His165 170 175 gcg gct ctc cat atc aat cct tac tat ggg aag acc tct atc gaaggg 576 Ala Ala Leu His Ile Asn Pro Tyr Tyr Gly Lys Thr Ser Ile Glu Gly180 185 190 ttg ata tct cat ttt gag gct gtc ctc cca atg ggt cca acc attatt 624 Leu Ile Ser His Phe Glu Ala Val Leu Pro Met Gly Pro Thr Ile Ile195 200 205 tac aat gtt cca tct agg act ggc cag gat att cct cct gca gttatt 672 Tyr Asn Val Pro Ser Arg Thr Gly Gln Asp Ile Pro Pro Ala Val Ile210 215 220 gag gct gtt tca agt ttc aca aac ttg gca ggt gtg aaa gaa tgtgtt 720 Glu Ala Val Ser Ser Phe Thr Asn Leu Ala Gly Val Lys Glu Cys Val225 230 235 240 gga cat gag agg gtt aag tgc tac act gac aaa ggt ata accata tgg 768 Gly His Glu Arg Val Lys Cys Tyr Thr Asp Lys Gly Ile Thr IleTrp 245 250 255 agt ggt aat gat gat gaa tgc cat gat tct agg tgg aaa tatggt gcc 816 Ser Gly Asn Asp Asp Glu Cys His Asp Ser Arg Trp Lys Tyr GlyAla 260 265 270 act gga gtt att tct gtg gct agc aac ctt att cct ggt ctcatg cac 864 Thr Gly Val Ile Ser Val Ala Ser Asn Leu Ile Pro Gly Leu MetHis 275 280 285 gat ctc atg tat gaa ggg gag aat aag acg cta aat gag aagctc ttt 912 Asp Leu Met Tyr Glu Gly Glu Asn Lys Thr Leu Asn Glu Lys LeuPhe 290 295 300 ccc ctg atg aaa tgg ttg ttt tgc cag cca aat cca att gctctc aac 960 Pro Leu Met Lys Trp Leu Phe Cys Gln Pro Asn Pro Ile Ala LeuAsn 305 310 315 320 act gcc ctg gct cag ctt gga gtg gta agg cct gtt ttcaga tta cca 1008 Thr Ala Leu Ala Gln Leu Gly Val Val Arg Pro Val Phe ArgLeu Pro 325 330 335 tat gta cct ctt cct ctt gaa aag agg gta gag ttt gtccga atc gtt 1056 Tyr Val Pro Leu Pro Leu Glu Lys Arg Val Glu Phe Val ArgIle Val 340 345 350 gaa tct att gga cgg gaa aac ttt gtg ggt gag aac gaggca cgg gtt 1104 Glu Ser Ile Gly Arg Glu Asn Phe Val Gly Glu Asn Glu AlaArg Val 355 360 365 ctt gac gac gat gat ttt gtg ttg gtc agt agg tac taa1143 Leu Asp Asp Asp Asp Phe Val Leu Val Ser Arg Tyr 370 375 380 8 380PRT Oryza sativa 8 Met Ala Ser Leu Leu Ile Ala Ser Thr Gly Gly Cys ProPro Pro Arg 1 5 10 15 Val Glu Gly Arg Arg Arg Pro Gly Thr Arg Ser GlyLeu Ala Arg Pro 20 25 30 Trp Pro Ala Ala Val Ala Ala Pro Ala Pro Leu LeuArg Ile Ser Arg 35 40 45 Gly Lys Phe Ala Leu Gln Ala Ile Thr Leu Asp AspTyr Leu Pro Met 50 55 60 Arg Ser Thr Glu Val Lys Asn Arg Thr Ser Thr AlaAsp Ile Thr Ser 65 70 75 80 Leu Arg Val Ile Thr Ala Val Lys Thr Pro TyrLeu Pro Asp Gly Arg 85 90 95 Phe Asp Leu Glu Ala Tyr Asp Ser Leu Ile AsnMet Gln Ile Asp Gly 100 105 110 Gly Ala Glu Gly Val Ile Val Gly Gly ThrThr Gly Glu Gly His Leu 115 120 125 Met Ser Trp Asp Glu His Ile Met LeuIle Gly His Thr Val Asn Cys 130 135 140 Phe Gly Ala Lys Val Lys Val ValGly Asn Thr Gly Ser Asn Ser Thr 145 150 155 160 Arg Glu Ala Ile His ValThr Glu Gln Gly Phe Ala Val Gly Met His 165 170 175 Ala Ala Leu His IleAsn Pro Tyr Tyr Gly Lys Thr Ser Ile Glu Gly 180 185 190 Leu Ile Ser HisPhe Glu Ala Val Leu Pro Met Gly Pro Thr Ile Ile 195 200 205 Tyr Asn ValPro Ser Arg Thr Gly Gln Asp Ile Pro Pro Ala Val Ile 210 215 220 Glu AlaVal Ser Ser Phe Thr Asn Leu Ala Gly Val Lys Glu Cys Val 225 230 235 240Gly His Glu Arg Val Lys Cys Tyr Thr Asp Lys Gly Ile Thr Ile Trp 245 250255 Ser Gly Asn Asp Asp Glu Cys His Asp Ser Arg Trp Lys Tyr Gly Ala 260265 270 Thr Gly Val Ile Ser Val Ala Ser Asn Leu Ile Pro Gly Leu Met His275 280 285 Asp Leu Met Tyr Glu Gly Glu Asn Lys Thr Leu Asn Glu Lys LeuPhe 290 295 300 Pro Leu Met Lys Trp Leu Phe Cys Gln Pro Asn Pro Ile AlaLeu Asn 305 310 315 320 Thr Ala Leu Ala Gln Leu Gly Val Val Arg Pro ValPhe Arg Leu Pro 325 330 335 Tyr Val Pro Leu Pro Leu Glu Lys Arg Val GluPhe Val Arg Ile Val 340 345 350 Glu Ser Ile Gly Arg Glu Asn Phe Val GlyGlu Asn Glu Ala Arg Val 355 360 365 Leu Asp Asp Asp Asp Phe Val Leu ValSer Arg Tyr 370 375 380 9 32 DNA Oryza sativa 9 gcctctcttg ttgagatactacctgtgttg cc 32 10 30 DNA Oryza sativa 10 gcaaatccct gctctgttacatgaatagcc 30 11 20 DNA Oryza sativa 11 gtaaaacgac ggccagtgag 20 12 19DNA Oryza sativa 12 ggaaacagct atgaccatg 19 13 21 DNA Oryza sativa 13tagggcgaat tgtgtgtacc g 21 14 30 DNA Oryza sativa 14 gctctagacaagatggcgtc gctgctgatc 30 15 28 DNA Oryza sativa 15 gcgagctcgt tagtacctactgaccaac 28

What is claimed is:
 1. An isolated DNA molecule for encoding the ricedihydrodipicolinate synthase which has the amino acid sequence shown inSEQ ID No. 2 of Sequence Listing.
 2. An isolated DNA molecule forencoding a protein having the amino acid sequence shown in SEQ ID No. 6of the Sequence Listing and having dihydrodipicolinate synthaseactivity.
 3. An isolated DNA molecule for encoding a protein having theamino acid sequence shown in SEQ ID No. 8 of the Sequence Listing andhaving dihydrodipicolinate synthase activity.